WO1991013078A1 - Biosynthese de carotenoïdes dans hotes transformes par genetique - Google Patents

Biosynthese de carotenoïdes dans hotes transformes par genetique Download PDF

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WO1991013078A1
WO1991013078A1 PCT/US1991/001458 US9101458W WO9113078A1 WO 1991013078 A1 WO1991013078 A1 WO 1991013078A1 US 9101458 W US9101458 W US 9101458W WO 9113078 A1 WO9113078 A1 WO 9113078A1
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plasmid
dna
phytoene
gene
enzyme
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PCT/US1991/001458
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Rodney Lee Ausich
Friedhelm Luetke Brinkhaus
Indrani Mukharji
John Houston Proffitt
James Gregory Yarger
Huei-Che Bill Yen
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Amoco Corporation
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Priority to DE69132769T priority Critical patent/DE69132769T2/de
Priority to EP91905713A priority patent/EP0471056B1/fr
Priority to DK91905713T priority patent/DK0471056T3/da
Priority to CA002055447A priority patent/CA2055447C/fr
Publication of WO1991013078A1 publication Critical patent/WO1991013078A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/847—Erwinia

Definitions

  • the present invention relates to carotenoid biosynthesis. More specifically, this invention relates to the isolation, characterization and expression of the six Erwinia herbicola genes encoding the enzymes geranylgeranyl pyrophosphate (GGPP) synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase and zeaxanthin glycosylase that catalyze the formation of geranylgeranyl pyrophosphate and the carotenoids phytoene, lycopene, 0-carotene, zeaxanthin and zeaxanthin diglucoside, respectively, each formed product (through zeaxanthin) being an immediate precursor for the next-named product.
  • GGPP geranylgeranyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • phytoene synthase phytoene dehydrogena
  • the invention also relates to methods for expression of these Erwinia herbicola enzyme genes in pro aryote hosts such as Escherichia coli and Aqrobacterium tumefaciens. in eukaryote hosts such as yeasts like Saccharomyces cerevisiae and higher plants such as alfalfa and tobacco, as well as to methods for preparation of GGPP and those carotenoids.
  • pro aryote hosts such as Escherichia coli and Aqrobacterium tumefaciens.
  • eukaryote hosts such as yeasts like Saccharomyces cerevisiae and higher plants such as alfalfa and tobacco, as well as to methods for preparation of GGPP and those carotenoids.
  • Carotenoids are 40-carbon (C A0 ) terpenoids consisting generally of eight isoprene (C 5 ) units joined together. Linking of the units is reversed at the center of the molecule. Trivial names and abbreviations will be used throughout this disclosure, with IUPAC-recommended semisystematic names given in parentheses after first mention of each name. Carotenoids are pigments with a variety of applications.
  • Phytoene (7,8,11,12,7• ,8• ,11' ,12' - ⁇ octahydro- ⁇ , ⁇ -carotene) is the first carotenoid in the carotenoid biosynthesis pathway and is produced by the dimerization of a 20-carbon atom precursor, geranylgeranyl pyrophosphate (GGPP) .
  • GGPP geranylgeranyl pyrophosphate
  • Phytoene has useful applications in treating skin disorders (U.S. Patent No. 4,642,318) and is itself a precursor for colored carotenoids. Aside from certain mutant organisms, such as Phvcomyces blakesleeanus carB, no current methods are available for producing phytoene via any biological process.
  • the red carotenoid lycopene ( ⁇ is the next carotenoid produced of the phytoene in the pathway.
  • lycopene imparts the characteristic red. color to ripe tomatoes.
  • Lycopene has utility as a food colorant. It is also an intermediate in the biosynthesis of other carotenoids in some bacteria, fungi and green plants.
  • Lycopene is prepared biosynthetically from phytoene through four sequential dehydrogenation reactions by the removal of eight atoms of hydrogen.
  • the enzymes that remove hydrogen from phytoene are phytoene dehydrogenases.
  • One or more phytoene dehydrogenases can be used to convert phytoene to lycopene and dehydrogenated derivatives of phytoene intermediate to lycopene are also known.
  • some strains of Rhodobacter sphaeroides contain a phytoene dehydrogenase that removes six atoms of hydrogen from phytoene to produce neurosporene.
  • phytoene dehydrogenase-4H The Rhodobacter phytoene dehydrogenase that removes three moles of hydrogen from each mole of phytoene will be hereinafter referred to as phytoene dehydrogenase-3H so that the distinctions 5 between the two enzymes discussed herein can be readily maintained.
  • Lycopene is an intermediate in the biosynthesis of carotenoids in some bacteria, fungi, and all green plants.
  • Carotenoid-specific genes that 10 can be used for synthesis of lycopene from the ubiquitous precursor farnesyl pyrophosphate include those for the enzymes GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H.
  • Beta-carotene is the third carotenoid produced 15 in the Erwinia herbicola carotenoid biosynthesis pathway. It is also synthesized by a number of bacteria, fungi, and most green plants.
  • Beta-carotene has utility as a colorant for margarine and butter, as a source for vitamin A 20 production, and has recently been implicated as having preventative effects against certain kinds of cancers.
  • Beta-carotene appears to have a protective effect without its conversion to vitamin A. Ziegler, Amer. Instit. Nutr.. publication 022/3166/89, 116 (1989). 30 Beta-carotene is produced by the cyclization of unsaturated carotenoids in a procedure not yet well understood. Bramley et al. In Current Topics in Cellular Regulation 29:291,297 (1988).
  • beta-carotene Current methods for commercial production of beta-carotene include isolation from carrots, chemical synthesis [Isler et al.. United States Patent 2,917,539 (1959) ] and microbial production by hoanephora trispora [Zajic, United States Patents 2,959,521 (1960) and 3,128,236 (1964)].
  • Zeaxanthin ( ⁇ , ⁇ -carotene-3,3'-diol) is the fourth carotenoid produced in the Erwinia herbicola carotenoid biosynthesis pathway. Zeaxanthin, a yellow pigment, is currently used as a colorant in the poultry industry.
  • zeaxanthin production Chemical synthesis methods for zeaxanthin production exist, but these are inefficient and not commercially competitive with the existing biomass sources.
  • the commercial sources for zeaxanthin are corn grain, corn gluten meal and marigold petals.
  • the level of zeaxanthin in corn kernels averages about 0.001 percent (dry weight) and the level in corn gluten meal averages about 0.01 percent (dry weight) . All these sources are characterized by low and inconsistent production levels.
  • Zeaxanthin diglucoside the fifth carotenoid produced in the Erwinia herbicola carotenoid biosynthesis pathway, is also useful as a food colorant and has a yellow color similar to that of zeaxanthin.
  • Carotenoids are synthesized in a variety of bacteria, fungi, algae, and higher plants. At the present time only a few plants are widely used for commercial carotenoid production. However, the productivity of carotenoid synthesis in these plants is relatively low and the resulting carotenoids are expensively produced.
  • Erwinia herbicola An organism capable of carotenoid synthesis and a potential source of genes for such an endeavor is Erwinia herbicola. which is believed to carry putative genes for carotenoid production on a plasmid (Thiry, J. Gen. Microbiol.. 130:1623 (1984)) or chromosomally (Perry et al., J. Bacteriol.. 168:607 (1986)). Erwinia herbicola is a genus of Gram-negative bacteria of the ENTEROBACTERIACEAE family, which are facultative anaerobes.
  • EP 0 393 690 Al reports use of DNA from Erwinia uredovora 20D3 (ATCC 19321) for preparing carotenoid molecules.
  • the present invention utilizes DNA from Erwinia herbicola EHO-10 (AT 39368) for preparation of carotenoid molecules and the enzymes used in their synthesis.
  • Erwinia herbicola EHO-10 used herein is also referred to as Escherichia vulneris.
  • the genus is commonly divided into three groups. Of the three, the Herbicola group includes species (e.g. E.
  • herbicola which typically form yellow pigments that have now been found to be carotenoids. These bacteria exist as saprotrophs on plant surfaces and as secondary organisms in lesions caused by many plant pathogens. They can also be found in soil, water and as opportunistic pathogens in animals, including man. A precise organismic function has yet to be ascribed to the pigment(s) produced by Erwinia herbicola. Perry et al., J. Bacteriol.. 168:607 (1986) , showed that the genes coding for the production of a then unknown yellow pigment lie within an approximately 13-kilobase (kb) sequence coding for at least seven polypeptides, and that the expression of the yellow pigment is cyclic AMP mediated.
  • kb 13-kilobase
  • E. coli and S. cerevisiae are commonly used for expressing foreign genes, but to optimize yields and minimize technical maintenance procedures, it would be preferable to utilize a higher plant species.
  • One aspect contemplated by this invention is an isolated DNA segment comprising a nucleotide sequence that defines a structural gene or variant DNA thereof capable of expressing each of the Erwinia herbicola genes for GGPP synthase (including a DNA analog) , phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase and zeaxanthin glycosylase in biologically active form.
  • Another aspect of this invention is a recombinant DNA molecule comprising a vector operatively linked to an above exogenous DNA segment isolated from Erwinia herbicola or a variant DNA.
  • This exogenous DNA segment defines a structural gene capable of expressing any one of the above Erwinia herbicola enzymes.
  • a promoter suitable for driving the expression of the enzyme in a compatible host organism is also included.
  • Exemplary, particularly preferred vectors include plasmids pARC417BH, pARC489B, pARC489D, pARC285, pARC140N, pARC145G, pARC496A, pARC146D, pATC228, pATC1616, pARC1509, pARC1510, pARC1520, pARC404BH, pARC406BH, pARC145H and pARC2019.
  • a further aspect of this invention is a method for preparing each of the above-mentioned E ⁇ . herbicola enzymes.
  • This method comprises initiating a culture, in a nutrient medium, of prokaryotic or eukaryotic host cells transformed with a recombinant DNA molecule containing an expression vector compatible with the cells.
  • This vector is operatively linked to an isolated exogenous Erwinia herbicola DNA segment or variant DNA that defines the structural gene for an above-mentioned particular enzyme.
  • the culture is maintained for a time period sufficient for the cells to express the enzyme.
  • Still another aspect contemplated by this invention is a method for producing GGPP, phytoene, lycopene, 3-carotene, zeaxanthin and/or zeaxanthin diglucoside that comprises initiating a culture in a nutrient medium of prokaryotic or eukaryotic host cells that provides the immediate precursor to the desired product, those prokaryotic or eukaryotic host cells being transformed with one or more recombinant DNA molecule(s) described herein that include a structural gene that can express an enzyme that converts the precursor to the product desired.
  • the culture is maintained for a time period sufficient for the host cells to express the enzyme and for the expressed enzyme to convert the provided precursor into product.
  • a recombinant DNA molecule contains an expression vector compatible with the host cells operatively linked to one or more exogenous Erwinia herbicola DNA or variant DNA segments comprising (i) a nucleotide base sequence corresponding to a sequence defining a structural gene for geranylgeranyl pyrophosphate synthase, (ii) a nucleotide base sequence corresponding to a sequence defining a structural gene for phytoene synthase, (iii) a nucleotide base sequence corresponding to a sequence defining a structural gene for phytoene dehydrogenase-4H, (iv) a nucleotide base sequence corresponding to a sequence defining a structural gene for lycopene cyclase, (v) a nucleotide base sequence corresponding to a sequence defining a structural gene for beta-carotene hydroxylase, and/or (vi) a nucleotide base sequence corresponding to a sequence defining
  • the culture is maintained for a time period sufficient for the cells to express the enzyme products of the desired structural genes (i) , (ii) , (iii) , (iv) , (v) and/or (vi) , and form a product that is desired.
  • Yet another aspect of this invention is a method of protecting a higher plant from the herbicide norflurazon.
  • a higher plant to be protected is transformed with a recombinant DNA molecule that encodes a structural gene for the Erwinia herbicola enzyme phytoene dehydrogenase-4H or a DNA variant thereof that encodes an enzyme exhibiting substantially the same biological activity.
  • the transformed plant is maintained for a time period sufficient for phytoene dehydrogenase-4H to be expressed, and transformed plant is treated with a herbicidal amount of norflurazon.
  • all of the recombinant DNA utilized in this invention is from Erwinia herbicola.
  • Another preferred embodiment of this invention is a recombinant DNA molecule as described above, wherein the promoter is Rec 7 for I__j_ coli. PGK. GAL 10 and GAL 1 for yeasts such as S. cerevisiae and CaMV 35S for higher plants.
  • a prokaryote such as Ej. coli.
  • a eukaryote for example yeast
  • Figure 1 is a flow diagram of the carotenoid biosynthesis pathway utilizing the Erwinia herbicola gene complement located in the plasmid pARC376.
  • Figure 2 in three sheets as Figure 2-1, Figure 2-2, and Figure 2-3 illustrates the nucleotide base sequences of certain preferred DNA segments of the structural gene for geranylgeranyl pyrophosphate (GGPP) synthase (SEQ ID NO: 1) .
  • the base sequences are shown conventionally from left to right and in the direction of 5' terminus to 3' terminus, using the single letter nucleotide base code.
  • the reading frame of the 5* end of the structural gene illustrated herein is indicated by placement of the deduced, amino acid residue sequence (SEQ ID NO: 2) of the protein for which it codes below the nucleotide sequence, such that the triple letter code for each amino acid residue is located directly below the three-base codon for each amino acid residue.
  • Numerals to the right of the DNA sequence indicate nucleotide base positions within the DNA sequence shown. All of the structural genes shown in the figures herein are similarly illustrated, with amino acid initiation position beginning here with the initial methionine residue (Met) at DNA position about 124 as shown.
  • Figure 3-2 and Figure 3-3 illustrates the DNA (SEQ ID NO: 3) and deduced amino acid residue (SEQ ID NO: 4) sequences of more preferred, heterologous structural genes of Erwinia herbicola GGPP synthase.
  • the expressed protein begins with the Met residue at about position 150 as shown and terminates within the Eco RV site (about 1153) in the DNA construct present in plasmid pARC489B, whereas the gene terminates at the Bal I site (about 1002) in the DNA construct present in plasmid pARC489D.
  • the short amino-terminal sequence MetAlaGluPhe (about 150-161) is a heterologous sequence from plasmid pARC306A, and is substituted for the native sequence from DNA position 124 to 150 shown in Figure 2. 5 Figure 4 shown in three sheets as Figure 4-1,
  • Figure 4-2 and Figure 4-3 illustrates the nucleotide (SEQ ID NO: 5) and amino acid (SEQ ID NO: 6) sequences of the structural gene for phytoene synthase.
  • the illustrated Bgl II restriction site shown at about position 8 is
  • Figure 5 schematically illustrates the plasmid pARC376 containing the full complement of enzyme genes, represented by capital letters, required for the
  • FIG. 6 is a schematic representation of the plasmid pARC306A, which contains the Rec 7 promoter.
  • This plasmid also has multiple cloning sites adjacent to the Rec 7 promoter and 5' and 3* transcription termination loops. Approximate positions of restriction enzyme sites are shown.
  • FIG. 7 illustrates schematically the plasmid pARC135, which contains the S. cerevisiae phosphoglyceric acid kinase (PGK) promoter operatively linked at the Bgl II site.
  • PGK phosphoglyceric acid kinase
  • Figure 8 shows a schematic representation of the vector pSOC713, including a partial restriction enzyme map.
  • Figure 9 is a schematic representation of plasmid pARC145B, which is a veast/E. coli shuttle vector for expression of introduced genes in yeast, including a partial restriction enzyme map.
  • Figure 10 is a schematic representation of the vector pARC145G, which is basically pARC145B above that contains the two preferred genes; i.e., GGPP synthase and phytoene synthase, each operatively linked at their 5* ends to the divergent promoters GAL 10 and GAL 1.
  • Phytoene synthase also has a PGK terminator at the 3 • end.
  • FIG 11 shown in four panels as Figure 11-1,
  • Figure 11-2, Figure 11-3 and Figure 11-4 illustrates the DNA (SEQ ID NO: 7) and deduced amino acid residue
  • SEQ ID NO: 8 sequences of the Erwinia herbicola structural gene for phytoene dehydrogenase-4H.
  • the Met codon (shown at position 7) corresponds to position
  • Figure 12 is a schematic representation of the vector pSOC925, including a partial restriction enzyme map.
  • Figure 13 is a schematic representation of plasmid pARC146, including a partial restriction enzyme map.
  • Figure 14 shows the vector pARC146D, including a partial restriction enzyme map.
  • Figure 15 shown in four panels as Figures 15- 1, Figures 15-2, Figure 15-3 and Figure 15-4 illustrates the DNA (SEQ ID NO: 9) and deduced amino acid residue (SEQ ID NO: 99) sequence of the Erwinia herbicola structural gene for phytoene dehydrogenase-4H present in plasmid pARC146D.
  • FIG 16 is a schematic representation of plasmid pATC228, including a partial restriction enzyme map.
  • Figure 17 illustrates the coding sequence and encoded transit peptide (SEQ ID NO: 98) DNA (SEQ ID NO: 10) sequence linked to the 5 1 end of the phytoene dehydrogenase-4H structural gene for transport of the phytoene dehydrogenase-4H enzyme into tobacco chloroplasts as well as other plant chloroplasts. Stars over nucleotide positions 69 and 72 in this sequence indicate G for T and G for A replacements utilized to introduce an Nar I site.
  • Figure 18 is a schematic representation of the about 15.6 kb plasmid pATC1616, including a partial restriction enzyme map.
  • Figure 19 shown in three panels as Figure 19- 1, Figure 19-2 and Figure 19-3 illustrates the DNA (SEQ ID NO: 11) and a deduced amino acid residue (SEQ ID NO: 13) sequences of the Erwinia herbicola structural gene for lycopene cyclase.
  • the Met codon (shown at position 19) corresponds to position 9002 on plasmid pARC376 in Figure 5.
  • the restriction sites Sph I and Bam HI were introduced at the 5 1 and 3' ends of the gene using PCR.
  • the changes in the sequence for the genetically engineered version of the gene (SEQ ID NO: 12) used for expression in yeast are shown in bold underneath the native sequence.
  • the native initiation GTG codon has been changed to an ATG codon.
  • the second amino acid residue, Arg was originally encoded by an AGG codon that was changed to a CGG codon, while retaining its coding for the Arg amino acid residue.
  • Figure 20 shown in three panels as Figure 20-1, Figure 20-2 and Figure 20-3 illustrates the DNA sequence of the native Erwinia herbicola DNA (SEQ ID NO: 14) segment containing the structural gene for beta-carotene hydroxylase, corresponding to a DNA segment from position 4886 to position 5861 of pARC376 shown in Figure 5.
  • the Met initiation codon is located at position 25 as shown, which corresponds to position 4991 in Figure 5.
  • FIG. 21-1, Figure 21-2 and Figure 21-3 illustrates the DNA (SEQ ID NO: 15) and deduced amino acid residue (SEQ ID NO: 16) sequences of the engineered Erwinia herbicola structural gene for beta-carotene hydroxylase.
  • the Met codon (shown at position 25) corresponds to position 4991 on plasmid pARC376 in Figure 5.
  • the restriction sites Nco I and Sma I were introduced at the 5' and 3* ends of the gene as described in Example 21.
  • the changes in the sequence for the genetically engineered version of the gene are shown in bold.
  • the native second and third amino acid residues have been changed from -Leu-Val- to -Val-Leu-.
  • Figure 22 is a schematic representation of plasmid pARC300E, including a partial restriction enzyme map showing restriction sites present only once.
  • Figure 23 is a schematic representation of plasmid pARC300M, including a partial restriction enzyme map showing restriction sites present only once.
  • Figure 24 is a schematic representation of plasmid pARC300T, including a partial restriction enzyme map showing restriction sites present only once.
  • Figure 25 shown in three panels as Figure 25- 1, Figure 25-2 and Figure 25-3 illustrates the DNA (SEQ ID NO: 97) and deduced amino acid residue (SEQ ID NO: 17) sequences of the engineered Erwinia herbicola structural gene for zeaxanthin glycosylase.
  • Expression The combination of intracellular processes, including transcription and translation undergone by a structural gene to produce a polypeptide.
  • Expression vector A DNA sequence that forms control elements that regulate expression of structural genes when operatively linked to those genes.
  • a structural gene is covalently bonded in correct reading frame to another DNA (or RNA as appropriate) segment, such as to an expression vector so that the structural gene is under the control of the expression vector.
  • Promoter A recognition site on a DNA sequence or group of DNA sequences that provide an expression control element for a gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.
  • Recombinant DNA molecule A hybrid DNA sequence comprising at least two nucleotide sequences not normally found together in nature.
  • Structural gene A DNA sequence that is expressed as a polypeptide, i.e., an amino acid residue sequence.
  • Vector A DNA molecule capable of replication in a cell and/or to which another DNA segment can be operatively linked so as to bring about replication of the attached segment.
  • a plasmid is an exemplary vector.
  • carotenoids are present in all photosynthetic organisms, where they are an essential part of the photosynthetic apparatus.
  • Mevalonic acid the first specific precursor of all the terpenoids is formed from acetyl-CoA via HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) , and is itself converted to isopentenyl pyrophosphate (IPP) , the universal isoprene unit.
  • IPP isopentenyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • the initial step is the condensation of farnesyl pyrophosphate (FPP) and isopentenyl pyrophosphate (IPP) to form geranylgeranyl pyrophosphate (GGPP) .
  • FPP farnesyl pyrophosphate
  • IPP isopentenyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • This first step is immediately followed by a tail-to-tail dimerization of GGPP, catalyzed by the enzyme phytoene synthase, to form phytoene.
  • This pathway thus differs from the pathway reported in published European Application 0 393 690 wherein GGPP is said to form prephytoene pyrophosphate (a cyclopropylene-containing molecule) that thereafter forms phytoene.
  • Lycopene has now been found to be the second carotenoid produced in Erwinia herbicola.
  • the third carotenoid produced by Erwinia herbicola results from the cyclization of lycopene to form beta-carotene, by the enzyme lycopene cyclase.
  • the fourth carotenoid in the Erwinia herbicola pathway is zeaxanthin that is produced from beta-carotene.
  • the fifth carotenoid, zeaxanthin diglucoside is produced from zeaxanthin.
  • the present invention relates to these steps in the carotenoid biosynthesis pathway, the methods of isolating the Erwinia herbicola genes encoding carotenoid biosynthesis enzymes of the pathway and to the adaptation of this pathway by recombinant DNA technology to achieve methods and capabilities of GGPP and carotenoid production, particularly in host organisms that do not otherwise synthesize those materials, but in relatively small amounts or in specialized locations.
  • the disclosure below provides a detailed description of the isolation of carotenoid synthesis genes from Erwinia herbicola. modification of these genes by genetic engineering, and their insertion into compatible plasmids suitable for cloning and expression in E. coli. yeasts, fungi and higher plants. Also disclosed are methods for preparation of the appropriate enzymes and the methods for GGPP and carotenoid production in these various hosts.
  • Plasmid constructs are exemplified for several host systems. However, similar constructs utilizing the genes of this invention are available for virtually any host system as is well known in the art.
  • a structural gene or isolated, purified DNA segment of this invention is often referred to as a restriction fragment bounded by two restriction endonuclease sites and containing a recited number of base pairs.
  • a structural gene of this invention is also defined to include a sequence shown in a figure plus variants of such genes (described hereinafter) , that hybridize non-randomly with a gene shown in the figure under stringency conditions as described hereinafter.
  • Each contemplated gene includes a recited non-randomly-hybridizable variant DNA sequence, encodes a particular enzyme and also produces biologically active molecules of the encoded enzymes when suitably transfected into and expressed in an appropriate host.
  • Polynucleotide hybridization is a function of sequence identity (homology) , G+C content of the sequence, buffer salt content, sequence length and duplex melt temperature (T m ) among other variables. See, Maniatis et al., Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982), page 388.
  • the buffer salt concentration and temperature provide useful variables for assessing sequence identity (homology) by hybridization techniques. For example, where there is at least 90 percent homology, hybridization is carried out at 68 ⁇ C in a buffer salt such as 6XSCC diluted from 20XSSC [Maniatis et al., above, at page 447].
  • the buffer salt utilized for final Southern blot washes can be used at a low concentration, e.g., 0.1XSSC and at a relatively high temperature, e.g. 68 ° C, and two sequences will form a hybrid duplex (hybridize) .
  • Use of the above hybridization and washing conditions together are defined as conditions of high stringency or highly stringent conditions.
  • Moderately high stringency conditions can be utilized for hybridization where two sequences share at least about 80 percent homology.
  • hybridization is carried out using 6XSSC at a temperature of about 50-55°C.
  • a final wash salt concentration of about 1-3XSSC and at a temperature of about 60-68°C are used.
  • These hybridization and washing conditions define moderately high stringency conditions.
  • Low stringency conditions can be utilized for hybridization where two sequences share at least 40 percent homology.
  • hybridization carried out using 6XSSC at a temperature of about 40-50°C, and a final wash buffer salt concentration of about 6XSSC used at a temperature of about 40-60 ⁇ C effect non- random hybridization.
  • These hybridization and washing conditions define low stringency conditions.
  • An isolated DNA or RNA segment that contains a nucleotide sequence that is at least 80 percent, and more preferably at least 90 percent identical to a DNA sequence for gene shown in a figure is contemplated by this invention.
  • a nucleotide sequence when present in a host cell as part of a plasmid or integrated into the host genome as described herein, that also hybridizes non-randomly under at least moderately high stringency conditions, and encodes and expresses a biologically active enzyme is contemplated herein as a variant DNA of an illustrated sequence that exhibits substantially the same biological activity.
  • amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid sequence of the structural gene that codes for the protein.
  • a structural gene can be defined in terms of the amino acid residue sequence; i.e., protein or polypeptide, for which it codes.
  • RNA sequences can be prepared that encode the same amino acid residue sequences, but that are sufficiently different from a before-discussed gene sequence that the two sequences do not hybridize at high stringency, but do hybridize at moderately high stringency.
  • allelic variants of a structural gene can exist in other Erwinia herbicola strains that are also useful, but form hybrid duplex molecules only at moderately high stringency.
  • a DNA or RNA sequence that (1) encodes an enzyme molecule exhibiting substantially the same biological activity as an enzyme molecule expressed by a DNA sequence of a figure, (2) hybridizes with a DNA sequence of one of those figures at least at moderately high stringency and (3) shares at least 80 percent, and more preferably at least 90 percent, identity with a DNA sequence of those figures is defined as a variant DNA sequence.
  • a DNA variant or variant DNA sequence is defined as including an RNA sequence.
  • the reported E_ s _ uredovora DNA sequences and the E ⁇ _ herbicola DNAs discussed herein are distinguishable, and genes that produce similar enzymes are not variants.
  • Variant DNA molecules that encode and can express a desired GGPP or carotenoid enzyme can be obtained from other organisms using hybridization and functionality selection criteria discussed herein. For example, a microorganism, fungus, alga, or higher plant that is known or can be shown to produce a carotenoid is utilized as a DNA source.
  • the total DNA of the selected organism is obtained and a genomic library is constructed in a ⁇ phage such as ⁇ gtll using the protocols discussed in Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982) at pages 270-294.
  • the phage library is then screened under standard protocols using a radiolabeled, nick- translated DNA probe having a sequence of the Erwinia herbicola DNA of the figures, and the before-discussed moderate or high stringency hybridization conditions.
  • that structural gene DNA segment can be obtained, sequenced, engineered for expression in an appropriate recombinant molecule and shown to produce a biologically active enzyme as is discussed elsewhere herein and other techniques and protocols that are well known to workers skilled in molecular biology. That a DNA sequence variant encodes a "biologically active" enzyme or an enzyme that has "substantially the same biological activity" is determined by whether the variant DNA sequence produces an enzyme as discussed herein.
  • a DNA variant sequence that expresses GGPP synthase or a carotenoid biosynthesis enzyme that converts a provided precursor substrate molecule into a desired product is defined as biologically active. Expression of a biologically active enzyme from a variant DNA sequence can be assayed by the production of the desired product.
  • a DNA segment of the invention thus includes a
  • that DNA segment includes a DNA sequence that encodes the enzyme in a DNA segment separate from any other carotenoid-forming enzyme encoding sequences. More preferably, a DNA segment contains the Erwinia herbicola GGPP synthase or carotenoid biosynthesis enzyme structural gene, and is free from a functional gene whose expression product consumes the desired carotenoid, or otherwise inhibits carotenoid production.
  • the plasmid pARC376 contains an approximately 13 kb chromosomal DNA fragment isolated by Perry et al. J. Bacteriol., 168:607 (1986) from the bacterium Erwinia herbicola EHO-10 (Escherichia vulneris; ATCC 39368) that when transferred into the bacterium E. coli causes the E. coli cells to produce a yellow pigment. Plasmid pARC376 was referred to by those authors as plasmid pPL376. A restriction map of the pARC376 plasmid is shown in Figure 5.
  • the structural genes in the plasmid responsible for pigment production are present on a DNA fragment of about 7900 base pairs (bp) that is bounded by the restriction sites Pst I (at about position 4886) and Bgl II (at about position 12349) shown in Figure 5.
  • Pst I at about position 4886
  • Bgl II at about position 12349
  • E. coli cells and all cells contemplated as hosts herein, naturally synthesize the isoprenoid intermediate farnesyl pyrophosphate (FPP) .
  • the genes for geranylgeranyl pyrophosphate (GGPP) synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase, and zeaxanthin glycosylase are located in the approximately 7900 bp DNA fragment in pARC376.
  • E. coli cells that are transformed with the plasmid pARC376 are able to convert some of the endogenous FPP into carotenoids by utilizing the enzymes encoded on the plasmid.
  • An isolated, purified DNA segment comprising a nucleotide sequence of at least 850 base pairs that define a structural gene for the Erwinia herbicola enzyme GGPP synthase and its DNA variants is one structural gene contemplated by this invention.
  • a typical, useful DNA segment contains about 850 to about 1150 base pairs, whereas a more preferred DNA segment contains about 850 to about 1000 base pairs.
  • the native sequence includes about 924 bp. Larger DNA segments are also contemplated and are discussed hereinafter.
  • FIG. 5 An approximately 1153 bp fragment that extends from the Bgl II (about 12349) site to the Eco RV (about 11196) site of plasmid pARC376 is shown in Figure 5.
  • a preferred structural gene for GGPP synthase is within the about 1153 bp Bgl II to Eco RV restriction fragment shown in Figure 5 and contains the previously mentioned native structural gene of about 924 bp. This structural gene is within the approximately 1030 bp Nco I-Eco RV restriction fragment of plasmid pARC417BH.
  • GGPP synthase structural gene of Figure 3 from which the 3' Bal I-Eco RV fragment was removed provided the most active GGPP synthase found.
  • This structural gene of about 850 bp is within the approximately 1000 bp Nco I-Pvu II restriction fragment of pARC489D. This GGPP synthase gene is most preferred herein. Details of the above work are described hereinafter.
  • the DNA sequence 1 from ⁇ . uredovora in EP 0 393 690 is said there to encode the gene for converting prephytoene pyrophosphate to phytoene.
  • the DNA sequence of that European application has about 59 percent identity with the GGPP synthase illustrated herein. That E. uredovora DNA sequence 1 is an analog of the before- discussed GGPP synthase gene, and can also be used herein for preparing GGPP.
  • recombinant DNA molecules comprising a vector operatively linked to an exogenous DNA segment defining a structural gene capable of expressing the enzyme GGPP synthase, as described above, and a promoter suitable for driving the expression of the gene encoding the enzyme in a compatible host organism.
  • the vector and promoter are as described elsewhere herein.
  • Particularly preferred plasmid vectors include pARC417BH, pARC489B, pARC489D and pARC145G.
  • Phytoene Synthase Gene and Plasmid Construct a DNA segments An isolated, purified DNA segment comprising a nucleotide sequence of at least about 927 base pairs that define a structural gene for the Erwinia herbicola enzyme phytoene synthase and its DNA variants is also contemplated in this invention by producing phytoene from GGPP.
  • This structural gene typically contains about 1000 to about 1250 bp including the 927 bp of the native sequence, but can also contain a greater number as discussed hereinafter.
  • the structural gene for phytoene synthase lies between positions 6383 and 5457 of plasmid pARC376 ( Figure 5) .
  • a phytoene synthase gene useful herein at least includes a sequence shown in Figure 4.
  • the structural gene also includes an upstream sequence shown in Figure 4 from about position 8 (Bgl II site) to about position 15 (Nco I site) .
  • a preferred phytoene synthase gene is within the about 1112 bp Nco I-Eco RI fragment of plasmid pARC285. Also included within that about 1112 bp segment is the approximately 1040 bp Nco I-Bam HI fragment that also encodes the desired structural gene.
  • the most preferred structural gene includes a nucleotide base sequence in Figure 4 from about base 8 to about base 15 as well as from about base 841 to about base 1040, and contains about 1090 bp.
  • This most preferred gene is contained in the approximately 1176 base pair sequence of the Hpa I to Bam HI restriction sites and approximately 1238 bp Pvu II-Eco RI fragments present in the plasmid pARC140N, as well as in the approximately 1088 bp sequence of the Bgl II-Eco RI fragment of plasmid pARC140R.
  • a recombinant DNA molecule comprising a vector operatively linked to an exogenous DNA segment defining a structural gene capable of expressing the enzyme phytoene synthase, as discussed above, and a promoter suitable for driving the expression of the gene in a compatible host organism, is also useful in this invention.
  • the vector and promoter of this recombinant molecule are also as are discussed herein.
  • Particularly preferred plasmid vectors include pARC285, pARC140N, and pARC145G.
  • Another DNA segment of this invention is an isolated DNA segment comprising a nucleotide sequence that contains at least about 1470 bp that includes a sequence defining a structural gene capable of expressing the Erwinia herbicola enzyme phytoene dehydrogenase-4H and its DNA variants.
  • Phytoene dehydrogenase-4H converts phytoene to lycopene.
  • This phytoene dehydrogenase-4H enzyme has a molecular mass of about 51,000 daltons.
  • the native phytoene dehydrogenase-4H structural gene contains about 1470 bp and is located between positions 7849 and 6380 of plasmid pARC376.
  • a typical, useful DNA segment contains about 1500 base pairs and lies within the approximately 1891 bp Ava I (8231) to Nco I (6342) DNA fragment from pARC376 illustrated in Figure 5. Larger DNA segments are also contemplated, as discussed hereinafter.
  • a preferred DNA segment includes a nucleotide base sequence shown in Figure 11 from about base 15 to about base 1470.
  • Particularly preferred DNA segments include the bases between the engineered Nco I site at about position 5 of Figure 11-1 (the initial Met residue) and about position 1470 of Figure 11-4, and is present in the approximately 1505 bp Nco I-Nco I restriction fragment (Nco I fragment) of plasmid pARC496A, the approximately 1508 bp Sal I-Sal I restriction fragment (Sal I fragment) of plasmid pARC146D, and the approximately 1506 bp Sph I-Nco I fragment present in plasmid pATC228.
  • the sequence of the about 1508 bp Sal I fragment is illustrated in Figure 15.
  • a still further particularly preferred DNA segment is the approximately 2450 bp Xba I-Xba I fragment present in plasmid pATC1616.
  • This fragment contains an approximately 1683 bp portion that encodes a chloroplast transit peptide of tobacco ribulose bis- phosphate carboxylase-oxygenase (hereinafter referred to as a chloroplast transit peptide) (about 177 bp) operatively linked in frame to the 5 1 end of the above Sph I-Nco I about 1506 bp phytoene dehydrogenase-4H gene. That approximately 1683 bp fragment is flanked at its 5 1 end by an about 450 bp CaMV 35S promoter sequence and at its 3 1 end by an about 300 bp NOS polyadenylation sequence.
  • This DNA segment can be used for expression of phytoene dehydrogenase-4H in higher plants and transport of the expressed phytoene dehydrogenase-4H into chloroplasts such as those of tobacco.
  • Infection of a higher plant such as tobacco with A. tumefaciens containing plasmid pATC1616 caused genomic incorporation of DNA for the promoter, transit peptide- phytoene dehydrogenase-4H and NOS sequence, and makes the resultant plants resistant to the herbicide norflurazon.
  • restriction fragments having the same restriction enzyme cleavage sequence at both the 5' and 3' ends are sometimes referred to herein by reference to a single restriction enzyme.
  • the approximately 1505 bp Nco I-Nco I restriction fragment referred to above can also be referred to herein as an approximately 1505 bp Nco I fragment.
  • the approximately 1508 bp Sal I-Sal I fragment can be referred to as the approximately 1508 bp Sal I fragment
  • the approximately 2450 bp Xba I-Xba I fragment can be referred to as the approximately 2450 bp Xba I fragment.
  • a recombinant DNA molecule comprising a vector operatively linked to an exogenous DNA segment defining a structural gene capable of expressing the enzyme phytoene dehydrogenase-4H and a promoter suitable for driving the expression of the enzyme in a compatible host organism is also contemplated by this invention.
  • the structural gene has a nucleotide base sequence described above.
  • Particularly preferred plasmids include pARC496A, pARC146D, pATC228 and pATC1616.
  • an isolated DNA segment comprising a nucleotide sequence that contains at least about 1125 base pairs that includes a sequence defining a structural gene capable of expressing the Erwinia herbicola enzyme lycopene cyclase and its DNA variants.
  • This lycopene cyclase enzyme has a molecular mass of about 39,000 daltons and converts lycopene to 0-carotene.
  • a typical, useful DNA segment contains at least about 1125 base pairs and preferably at least about 1150 base pairs and lies within the approximately 1548 bp Sal I (9340) to Pst I (7792) DNA fragment from plasmid pARC376 illustrated in Figure 5.
  • the native Erwinia herbicola structural gene for lycopene cyclase contains about 1125 base paris and is located between positions 9002 and 7878 of plasmid pARC376. Larger DNA segments are also contemplated, as discussed hereinafter.
  • a preferred DNA segment includes a nucleotide base sequence shown in Figure 19, panels 1 and 2, from about base 1 to about base 1222.
  • a more preferred sequence of about 1140 bp is present in the approximately 1142 bp Sph I-Bam HI restriction fragment of the plasmid pARC1509, and shown in Figure 19.
  • a still further particularly preferred DNA segment is an approximately 1319 bp Nco I-Bam HI fragment.
  • This fragment contains an approximately 177 bp portion that encodes a chloroplast transit peptide operatively linked in frame to the 5' end of the above Sph I-Bam HI 1142 bp lycopene cyclase gene.
  • This DNA segment can be used for expression of lycopene cyclase in higher plants and transport of the expressed lycopene cyclase into chloroplasts such as those of tobacco.
  • a recombinant DNA molecule comprising a vector operatively linked to an exogenous DNA segment defining a structural gene capable of expressing the enzyme lycopene cyclase and a promoter suitable for driving the expression of that enzyme in a compatible host organism, is also contemplated by this invention.
  • the structural gene has a nucleotide base sequence described above.
  • Particularly preferred plasmid vectors include pARC1510, pARC1520, pARC1509.
  • an isolated DNA segment comprising a nucleotide sequence that contains at least about 531 base pairs and more preferably about 878 base pairs, including a sequence defining a structural gene capable of expressing the Erwinia herbicola enzyme beta-carotene hydroxylase, an enzyme that synthesizes zeaxanthin, and DNA variants of that gene.
  • the native enzyme is encoded between positions 4991 and 5521 of plasmid pARC376.
  • nucleotide base sequence corresponds to the sequence in Figure 20 from about base 1 to about base 894 at the S a I site, and preferably from about base' 1 to about base 752. More preferably, the DNA segment utilized is that shown in Figure 21 from about base 25 to about base 897. The latter sequence constitutes the about 870 bp Nco I to Sma I DNA fragment contained in plasmid pARC406BH. A contemplated DNA segment lies within the (4991) to (5861) DNA segment of about 870 base pairs of plasmid pARC376 illustrated in Figure 5.
  • a still further particularly preferred DNA segment is an Xba I-Xba I fragment including about 1797 bp constituted by the following sequence of genes: (a) the about 450 bp CaMV 35S promoter, (b) the 177 bp sequence that encodes a chlorplast transit peptide operatively linked in frame to the 5 1 end of (c) the about 870 bp beta-carotene hydroxylase gene, and (d) the about 300 bp NOS polyadenylation sequence.
  • This Xba I fragment can be cloned into the Xba I site of plasmid pGA482, with the resulting plasmid being used to transform A ⁇ . " tumefaciens. The resulting, transformed A ⁇ . tumefaciens can then be used to transform higher plants such as tobacco wherein the transit peptide-1inked enzyme is expressed and transporated to chlorplasts for the production of zeaxanthin.
  • a recombinant DNA molecule comprising a vector operatively linked to an exogenous DNA segment defining a structural gene capable of expressing the Erwinia herbicola enzyme beta-carotene hydroxylase and a promoter suitable for driving the expression of that enzyme in a compatible host organism, is also contemplated by this invention.
  • the structural gene has a nucleotide base sequence described above.
  • Particularly preferred recombinant DNA molecules include E_j_ coli plasmid pARC404BH that contains the about 874 bp Nco I-Sma I fragment, E_i.
  • coli plasmid pARC406BH that includes that same restriction fragment driven by the Rec 7 promoter, and plasmid pARC145H designed for S_j_ cerevisiae expression of GAL 1- or GAL 10-driven or PGK-driven and URA 3-terminated beta- carotene hydroxylase, as well as expression of GGPP synthase and phytoene synthase driven by the GAL 1 and GAL 10 promoters. 7. Zeaxanthin Glycosylase Gene and Plasmid Construct a. DNA Segment
  • an isolated DNA segment comprising a nucleotide sequence that contains at least about 1200 base pairs, including a sequence defining a structural gene capable of expressing the Erwinia herbicola enzyme zeaxanthin glycosylase, an enzyme that synthesizes zeaxanthin diglucoside, and variant DNAs.
  • the native DNA sequence for Erwinia herbicola lies between positions 10232 and 9033 of plasmid pARC376.
  • a preferred DNA encoding zeaxanthin glycosylase is in the about 1390 bp Nde I- Ava I fragment of plasmid pARC2019.
  • the DNA sequence of the IL. herbicola structural gene is shown in Figure 25 (SEQ ID NO:97) .
  • a further particularly preferred DNA segment is in an Xba I-Xba I fragment including about 2127 bp constituted by the following sequence of genes: (a) the abouat 450 bp CaMV 35S promoter, (b) the 177 bp sequence that encodes a chloroplast transit peptide operatively linked in frame to the 5' end of (c) the about 1200 bp zeaxanthin glycosylase gene, and (d) the about 300 bp NOS polyadenylation sequence.
  • This Xba I fragment can be cloned into the Xba I site of plasmid pGA482, with the resulting plasmid being used to transform A ⁇ . tumefaciens.
  • the resulting, transformed A. tumefaciens can then be used to transform higher plants such as tobacco wherein the transit peptide- linked enzyme is expressed and transporated to chlorplasts for the production of zeaxanthin diglucoside.
  • a recombinant DNA molecule comprising a vector operatively linked to an exogenous DNA segment defining a structural gene capable of expressing the Erwinia herbicola enzyme zeaxanthin glycosylase and a promoter suitable for driving the expression of that enzyme in a compatible host organism, is also contemplated by this invention.
  • the structural gene has a nucleotide base sequence described above.
  • Particularly preferred recombinant DNA molecules include the E. coli plasmid pARC2019 that contains the about 1390 bp Nde I-Ava I fragment, an E. coli plasmid driven by the Rec 7 promoter, and a plasmid designed for S. cerevisiae expression of a GAL-1-. GAL-10- or PGK-driven zeaxanthin glycosylase.
  • DNA segments are noted as having a minimal length, as well as total overall lengths. That minimal length defines the length of a DNA segment having a sequence that encodes a particular protein enzyme.
  • isolated DNA segments and variants thereof can be prepared by in vitro mutagenesis, as described in the examples, that begin at the initial ATG codon for a gene and end at or just downstream of the stop codon for each gene.
  • a desired restriction site can be engineered at or upstream of the initiation codon, and at or downstream of the stop codon so that shorter structural genes than most of those discussed above can be prepared, excised and isolated.
  • a DNA segment of the invention can be 2,000-15,000 base pairs in length.
  • the maximum size of a recombinant DNA molecule, particularly an expression vector is governed mostly by convenience and the vector size that can be accommodated by a host cell, once all of the minimal DNA sequences required for replication and expression, when desired, are present. Minimal vector sizes are well known. Such long DNA segments are not preferred, but can be used.
  • Example 4b illustrates that a DNA segment of several thousand base pairs that contains the structural genes for GGPP synthase and phytoene synthase can be used to produce phytoene.
  • the same situation is true for phytoene dehydrogenase-4H production as is seen in Example 9b.
  • the DNA segment used in Example 9b contains structural genes for GGPP synthase, phytoene synthase and phytoene dehydrogenase- 4H, lycopene cyclase and the other structural genes for zeaxanthin preparation.
  • the gene for lycopene cyclase which utilizes lycopene, was impaired so that no functional lycopene cyclase was produced and lycopene accumulated.
  • Example 16b wherein the gene for -carotene hydroxylase originally present in plasmid pARC376 was made inoperative and /3-carotene was found to accumulate.
  • DNA segments that encode the before-described enzyme proteins can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc. , 103:3185 (1981). (The disclosures of the art cited herein are incorporated herein by reference.) Of course, by chemically synthesizing the coding sequence, any desired modifications can be made simply by substituting the appropriate bases for those encoding the native amino acid residue sequence. However, DNA segments including sequences discussed previously are preferred.
  • DNA segments containing structural genes encoding the enzyme proteins can be obtained from recombinant DNA molecules (plasmid vectors) containing those genes.
  • plasmid vectors plasmid vectors
  • the plasmid type recombinant DNA molecules pARC417BH, pARC489B, pARC489D, pARC285, and pARC140N each contain DNA sequences encoding different portions of the GGPP synthase and phytoene synthase proteins and together possess the entire sequence of DNA necessary for expression of either protein in biologically active form.
  • Plasmid pARC145G contains DNA segments encoding both enzymes.
  • the plasmid type recombinant DNA molecules pARC496A, pARC146D, pATC228 and pATC1616 each contain a DNA sequence encoding biologically active phytoene dehydrogenase-4H proteins.
  • the plasmid type recombinant DNA molecules pARC1509, pARC1510, and pARC1520 each contain a DNA sequence encoding biologically active lycopene cyclase proteins.
  • plasmid type recombinant DNA molecules pARC404BH, pARC406BH and pARC145H each contain a DNA sequence encoding biologically active beta-carotene hydroxylase proteins
  • plasmid pARC2019 contains a DNA sequence that encodes biologically active zeaxanthin glycosylase.
  • Plasmids pARC417BH, pARC489B, pARC489D, pARC285, pARC140N and pARC145G have been deposited pursuant to Budapest Treaty requirements with the
  • accession No. 40806 American Type Culture Collection 12301 Parklawn Drive, Rockville, MD 20852 on February 26, 1990 and were assigned the following respective accession numbers 40755, 40758, 40757, 40756, 40759, and 40753.
  • Plasmids pARC496A, pARC146D and pATC228 were deposited pursuant to Budapest Treaty requirements with the American Type Culture Collection, (ATCC) 12301 Parklawn Drive, Rockville, MD 20852 on May 11, 1990 and were assigned the following respective accession numbers 40803, 40801 and 40802. Plasmid pATC1616 was similarly deposited on May 15, 1990 and was assigned accession No. 40806.
  • Plasmids pARC1509, pARC1510, and pARC1520 were deposited pursuant to Budapest Treaty requirements with the American Type Culture Collection, (AT) 12301 Parklawn Drive, Rockville, MD 20852 on July 27, 1990 and were assigned the following respective accession numbers 40850, 40851 and 40852.
  • Plasmids pARC404BH, pARC406BH and pARC145H were similarly deposited on January 16, 1991, and were assigned ATCC accession numbers 40943, 40945, and 40944, respectively.
  • Plasmid pARC2019 was also deposited in accordance with the Budapest Treaty on February 13, 1991 and received ATCC accession number 40974.
  • a DNA segment that includes a DNA sequence encoding zeaxanthin glycosylase, beta-carotene hydroxylase, lycopene cyclase, phytoene dehydrogenase- 4H, GGPP synthase, and phytoene synthase can be prepared by excising and operatively linking appropriate restriction fragments from each of the above deposited plasmids using well known methods.
  • the DNA molecules of the present invention produced in this manner typically have cohesive termini, i.e., "overhanging" single-stranded portions that extend beyond the double-stranded portion of the molecule.
  • the presence of cohesive termini on the DNA molecules of the present invention is preferred, although molecules having blunt termini are also contemplated.
  • RNA Ribonucleic acid equivalents of the above described DNA segments are also contemplated.
  • a recombinant DNA molecule of the present invention can be produced by operatively linking a vector to a DNA segment of the present invention to form a plasmid such as those discussed and deposited herein. Particularly preferred recombinant DNA molecules are discussed in detail in the examples, hereafter.
  • Vectors capable of directing the expression of GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase, and/or zeaxanthin glycosylase genes are referred to herein as "expression vectors".
  • the expression vectors described above contain expression control elements including the promoter.
  • the polypeptide coding genes are operatively linked to the expression vector to allow the promoter sequence to direct RNA polymerase binding and expression of the desired polypeptide coding gene.
  • Useful in expressing the polypeptide coding gene are promoters which are inducible, viral, synthetic, constitutive as described by Poszkowski et al., EMBO J.. 3:2719 (1989) and Odell et al.. Nature, 313:810 (1985), and temporally regulated, spatially regulated, and spatiotemporally regulated as given in Chua et al.. Science. 244:174-181 (1989).
  • a vector useful in practicing the present invention is capable of directing the replication, and preferably also the expression (for an expression vector) of the polypeptide coding gene included in the DNA segment to which it is operatively linked.
  • a vector in one preferred embodiment, includes a prokaryotic replicon; i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell transformed therewith.
  • a prokaryotic replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell transformed therewith.
  • Those vectors that include a prokaryotic replicon can also include a prokaryotic promoter region capable of directing the expression of the GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase or zeaxanthin glycosylase genes in a host cell, such as E. coli.
  • Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing one or more convenient restriction sites for insertion of a DNA segment of the present invention.
  • Typical of such vector plasmids are pUC8, pUC9, and pBR329 available from Biorad Laboratories, (Richmond, CA) and pPL and pKK223-3 available from Pharmacia, Piscataway, N.J.
  • a particularly preferred promoter for use in prokaryotic cells such as E. coli is the Rec 7 promoter present in plasmid vectors pARC306A, pARC496A and pARC136, and inducible by exogenously supplied nalidixic acid.
  • Expression vectors compatible with eukaryotic cells are also contemplated herein. Such expression vectors can also be used to form the recombinant DNA molecules of the present invention.
  • Vectors for use in yeasts such as S. cerevisiae can be episomal or integrating, as is well known.
  • Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources.
  • such vectors contain one or more convenient restriction sites for insertion of the desired DNA segment and promoter sequences.
  • exemplary promoters for use in S. cerevisiae include the S. cerevisiae phosphoglyceric acid kinase (PGK) promoter and the divergent promoters GAL 10 and GAL 1.
  • PGK phosphoglyceric acid kinase
  • Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al. , Meth. in Enzymol.. 153:253-277 (1987).
  • Plasmid pCaMVCN (available from Pharmacia, Piscataway, NJ) includes the cauliflower mosaic virus CaMV 35S promoter. The introduction of genes into higher plants is discussed in greater detail hereinafter.
  • retroviral expression vectors to form the recombinant DNAs of the present invention is also contemplated.
  • “retroviral expression vector” refers to a DNA molecule that includes a promoter sequence derived from the long terminal repeat (LTR) region of a retrovirus genome.
  • the retroviral expression vector is preferably replication- incompetent in eukaryotic cells.
  • the construction and use of retroviral vectors has been described by Verma, PCT Publication No. W087/00551, and Cocking et al, Science. 236:1259-62 (1987).
  • the vector used to express the polypeptide coding gene includes a selection marker that is effective in a plant cell, preferably a drug resistance selection marker.
  • a drug resistance selection marker is the gene whose expression results in kanamycin resistance, i.e., the chimeric gene containing the nopaline synthase promoter, Tn5 neomycin phosphotransferase II and nopaline synthase 3' nontranslated region described by Rogers et al., in Methods For Plant Molecular Biology. A. eissbach and H. Weissbach, eds., Academic Press Inc. , San Diego, CA (1988) .
  • Another preferred marker is the assayable chloramphenicol acetyltransferase (cat) gene from the transposon Tn9.
  • cat chloramphenicol acetyltransferase
  • DNA segment to be inserted and to the vector DNA are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • synthetic linkers containing one or more restriction endonuclease sites can be used to join the DNA segment to the expression vector.
  • the synthetic linkers are attached to blunt-ended DNA segments by incubating the blunt-ended DNA segments with a large excess of synthetic linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying synthetic linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction endonuclease and ligated into an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the synthetic linker.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including New England BioLabs
  • RNA equivalents of the above described recombinant DNA molecules are also contemplated by the present invention.
  • Methods for introducing polypeptide coding genes into higher, multicelled plants include Agrobacterium-mediated plant transformation, protoplast transformation, gene transfer into pollen, injection into reproductive organs and injection into immature embryos.
  • Agrobacterium-mediated plant transformation protoplast transformation
  • gene transfer into pollen injection into reproductive organs
  • injection into immature embryos injection into immature embryos.
  • Each of these methods has distinct advantages and disadvantages.
  • one particular method of introducing genes into a particular plant species may not necessarily be the most effective for another plant species, but it is well known which methods are useful for a particular plant species.
  • Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast.
  • the use of Agrobacterium-mediated expression vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et al., Biotechnology. 3:629 (1985) and Rogers et al., Methods in Enzymology. 153:253-277 (1987). Further, the integration of the Ti-DNA is a relatively precise process resulting in few rearrangements.
  • the region of DNA to be transferred is defined by the border sequences, and intervening DNA is usually inserted into the plant genome as described by Spielmann et al. , Mol.
  • GGPP synthase using the genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase and zeaxanthin glycosylase to induce zeaxanthin and zeaxanthin diglucoside synthesis in the cytoplasm is a viable approach, even though carotenoids are not naturally produced in the cytoplasm.
  • Agrobacterium-mediated transformation of leaf disks and other tissues appears to be limited to plant species that Agrobacterium naturally infects. Thus, Agrobacterium-mediated transformation is most efficient in dicotyledonous plants. However, as mentioned above, the transformation of asparagus using Agrobacterium can also be achieved. See, for example, Bytebier, et al., Proc. Natl. Acad. Sci.. 84:5345 (1987).
  • Transformation of plant protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments. See, for example, Potrykus et al., Mol. Gen. Genet., 199:183 (1985); Lorz et al., Mol. Gen. Genet.. 199:178 (1985); From et al., Nature, 319:791 (1986); Uchimiya et al., Mol. Gen. Genet.. 204:204 (1986); Callis et al., Genes and Development, 1:1183 (1987); and Marcotte et al., Nature. 335:454 (1988).
  • Metal particles have been used to successfully transform corn cells and to produce fertile, stably transformed tobacco and soybean plants. Transformation of tissue explants eliminates the need for passage through a protoplast stage and thus speeds the production of transgenic plants.
  • DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al., Methods in Enzymology. 101:433 (1983); D. Hess, Intern Rev. Cvtol.. 107:367 (1987); Luo et al., Plant Mol. Biol. Reporter. 6:165 (1988).
  • Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al., Nature. 325:274 (1987).
  • DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus et al. , Theor. Appl. Genet. , 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo 1986, Butterworth, Stoneham, MA, pp. 27-54 (1986) .
  • transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et aL.Proc. Natl. Acad. Sci. U.S.A. , 80:4803 (1983).
  • This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth.
  • Transformant shoots that rooted in the presence of the selective agent to form plantlets are then transplanted to soil or other media to allow the production of roots.
  • These procedures vary depending upon the particular plant species employed, such variations being well known in the art.
  • the transformed elements are so manipulated as to permit them to mature into soil- or otherwise-cultivated plants, such as plants that are cultivated hydroponically or in other soil-free media such as lava rock, crushed coral, sphagnum moss and the like.
  • tissue culture procedures are also contemplated, for example, using Agrobacterium-mediated vectors to produce transgenic plants from seeds.
  • the transgenic plant is harvested to recover the carotenoid product.
  • This harvesting step can consist of harvesting the entire plant, or only the leaves, or roots of the plant. This step can either kill the plant or if only a non- essential portion of the transgenic plant is harvested can permit the remainder of the plant to continue to grow.
  • At least a portion of the transgenic plant is homogenized to produce a plant pulp using methods well known to one skilled in the art.
  • This homogenization can be done manually, by a machine, or by a chemical means as long as the transgenic plant portions are broken up into small pieces to produce a plant pulp.
  • This plant pulp consists of a mixture of the carotenoid of interest residual amounts of precursors, cellular particles and cytosol contents. This pulp can be dried and compressed into pellets or tablets and eaten or otherwise used to derive the benefits, or the pulp can be subjected to extraction procedures.
  • the carontenoid can be extracted from the plant pulp produced above to form a solution or suspension.
  • the extracting step can consist of soaking or immersing the plant pulp in a suitable solvent.
  • This suitable solvent is capable of dissolving or suspending the carotenoid present in the plant pulp to produce a carotenoid-containing solution or suspension.
  • Solvents useful for such an extraction process are well known to those skilled in the art and include water, several organic solvents and combinations thereof such as methanol, ethanol, isopropanol, acetone, acetonitrile, tetrahydrofuran (THF) , hexane, and chloroform.
  • a vegetable oil such as peanut, corn, soybean and similar oils can also be used for this extraction.
  • Isolation (harvesting) of carotenoids from bacteria, yeasts, fungi and other lower organisms is illustrated hereinafter using A. tumefaciens and E. coli.
  • cells transfected with structural genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase and zeaxanthin glycosylase, as desired are grown under suitable conditions for a period of time sufficient for a desired carotenoid to be synthesized.
  • the carotenoid-containing cells are then lysed chemically or mechanically, and the carotenoid is extracted from the lysed cells using a liquid organic solvent, as described before, to form a carotenoid-containing liquid solution or suspension.
  • the carotenoid is thereafter isolated from the liquid solution or suspension by usual means such as chromatography.
  • the carotenoid is isolated from the solution or suspension produced above using methods that are well known to those skilled in the art of carotenoid isolation. These methods include, but are not limited to, purification procedures based on solubility in various liquid media, chromatographic techniques such as column chromatography and the like.
  • the present invention also relates to host cells transformed with recombinant DNA molecules of the present invention, preferably recombinant DNA capable of expressing GGPP synthase and membrane-bound (or soluble) phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase and zeaxanthin glycosylase enzymes.
  • GGPP synthase and membrane-bound (or soluble) phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase and zeaxanthin glycosylase enzymes zeaxanthin glycosylase enzymes.
  • the host cells can be either prokaryotic or eukaryotic.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example the E. coli strain HB101, available from BRL Life Technologies, Inc., Gaithersburg, MD (BRL) .
  • Preferred eukaryotic host cells include yeast and plant cells or protoplasts, preferably cells from higher plants.
  • Preferred eukaryotic host cells include S. cerevisiae cells such as YPH499 obtained from Dr. Phillip Hieter, Johns Hopkins University, Baltimore, MD, discussed in Example 5.
  • Transformation of appropriate cell hosts with a recombinant DNA molecule of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al., Proc. Natl. Acad. Sci. USA, 69:2110 (1972); and Maniatis et al., Molecular Cloning,
  • Successfully transformed cells i.e., cells that contain a recombinant DNA molecule of the present invention
  • cells resulting from the introduction of a recombinant DNA of the present invention can be cloned to produce monoclonal colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the recombinant DNA using a method such as that described by Southern, J. Mol. Biol.. 98:503 (1975) or Berent et al., Biotech.. 3:208 (1985) .
  • cells successfully transformed with an expression vector may produce proteins displaying GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase or zeaxanthin glycosylase antigenicity.
  • Identifying successful transformation of E. coli in this invention is relatively easy for carotenoids, except phytoene.
  • Carotenoid-containing colonies formed are usually characterized by colored pigment formation. For example, beta-carotene, zeaxanthin and zeaxanthin diglucoside are yellow and lycopene is red.
  • a method for preparing a carotenoid biosynthesis enzyme comprises initiating a culture, in a nutrient medium, of transformed prokaryotic or eukaryotic host cells.
  • the host cells are transformed with a recombinant DNA molecule containing a compatible expression vector operatively linked to a before- described exogenous DNA segment that defines the structural gene for a carotenoid biosynthesis enzyme, as desired.
  • This invention further comprises cultures maintained for a time period sufficient for the host cells to express the carotenoid biosynthesis enzyme protein molecules, which proteins can be recovered in purified form if desired.
  • Nutrient media useful for culturing transformed host cells are well known in the art and can be obtained from several commercial sources. A further discussion of useful host cells and nutrient media are provided in the following section.
  • a further aspect contemplated is a method for preparing one carotenoid biosynthesis enzyme in the presence of either or all of the other carotenoid biosynthesis enzymes such as GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, /3-carotene hydroxylase and zeaxanthin glycosylase.
  • This method is substantially identical to the before- described method except that the host cells are also transformed with a compatible expression vector operatively linked to a before-described exogenous DNA segment that defines any or all of the structural genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, J-carotene hydroxylase and zeaxanthin glycosylase.
  • the transformed host cell can contain a single expression vector that contains one or more of the six structural genes.
  • the host can also be transformed with two expression vectors each containing a structural gene for one or more of the enzymes; e.g., one for at least beta-carotene hydroxylase or zeaxanthin glycosylase and another that contains at least one of the other four (or five) enzymes, or three expression vectors; e.g., one for at least beta- carotene hydroxylase or zeaxanthin glycosylase, and two others that each contain at least one of the other four (or five) enzymes.
  • An exemplary four expression vector transformation can also be used in which at least one expression vector contains the structural gene that encodes beta-carotene hydroxylase or zeaxanthin glycosylase, with each of the other vectors containing at least one structural gene for each of the other structural genes.
  • a host cell can also be transformed with five vectors; i.e., one expression vector that contains the gene encoding each one of the first four enzymes and another vector containing the last two genes.
  • a six-vector system can also be utilized for production of zeaxanthin glycosylase in which a host is transformed with one expression vector for each of the six named enzymes.
  • a carotenoid can be produced by a method that includes initiating a culture, in a nutrient medium, of prokaryotic or eukaryotic host cells that are transformed with a recombinant DNA molecule containing a compatible expression vector operatively linked to a before-described exogenous DNA segment that defines the structural gene for a carotenoid biosynthesis enzyme that converts an immediate precursor substrate molecule into the desired carotenoid, and which cells provide the immediate precursor molecule that is the substrate for the expressed carotenoid biosynthesis enzyme.
  • the cell culture is maintained for a time period sufficient for the transformed cells to produce (express) the desired carotenoid biosynthesis enzyme, and for that expressed enzyme to convert the provided immediate precursor substrate molecule into the desired carotenoid.
  • the produced carotenoid can thereafter be recovered as discussed herein.
  • the nutrient medium and in many cases the enzyme substrate that is the immediate precursor molecule
  • the initiated culture is the germinated seed, protoplast or even a grafted explant from a prior culture.
  • the required carotenoid biosynthesis enzymes are provided to the cells by transformation of those cells with one or more exogenous recombinant DNA molecules that encode and express the appropriate genes so that appropriate enzymes and precursor substrate molecules are provided to the cells.
  • an exogenous structural gene for GGPP synthase is required to produce GGPP from the ubiguitous precursor FPP, and an exogenous structural gene for phytoene synthase is required to convert GGPP (the immediate precursor substrate) into phytoene.
  • GGPP the immediate precursor substrate
  • at least two exogenous recombinant DNA molecules are needed to produce a carotenoid.
  • the above transformed cells must also be further transformed with an exogenous recombinant DNA molecule that codes for and expresses phytoene dehydrogenase-4H. Further transformation of such transformed cells is needed as each ensuing carotenoid illustrated in Figure 1 that is desired to be prepared.
  • the exogenous structural genes used for the transformation can reside in a single recombinant DNA molecule, or in a plurality of such recombinant molecules as is exemplified below.
  • the recombinant DNA molecule contains an expression system that comprises one or more expression vectors compatible with host cells operatively linked to an exogenous DNA segment that comprises (i) a nucleotide base sequence corresponding to a sequence defining a structural gene for GGPP synthase as discussed before, and (ii) a nucleotide base sequence corresponding to a sequence defining a structural gene for phytoene synthase as also discussed before.
  • a particularly preferred expression vector plasmid pARC145G contains structural genes for both GGPP synthase and phytoene synthase, and produces phytoene in S. cerevisiae.
  • the recombinant DNA molecule preferably contains an expression system that comprises one or more expression vectors compatible with host cells, operatively linked to an exogenous DNA segment that comprises (i) a nucleotide base sequence corresponding to a sequence, defining a structural gene for GGPP synthase, and (ii) a nucleotide base sequence corresponding to a sequence defining a structural gene for phytoene synthase, and (i ⁇ ) a nucleotide base sequence corresponding to the sequence defining a structural gene for phytoene dehydrogenase-4H.
  • an expression system that comprises one or more expression vectors compatible with host cells, operatively linked to an exogenous DNA segment that comprises (i) a nucleotide base sequence corresponding to a sequence, defining a structural gene for GGPP synthase, and (ii) a nucleotide base sequence corresponding to a sequence defining a structural gene for phytoene synthase, and (i ⁇ )
  • the structural genes for GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H are contained operatively linked in a single expression vector, preferably under the control of the same promoter.
  • two expression vectors are used, with the structural genes for GGPP synthase and phytoene synthase on one vector and the structural gene for phytoene dehydrogenase-4H on the other vector.
  • three expression vectors are used. Yeast and plants require a separate promoter for each gene, although the same promoter can be used for each gene.
  • Example 9b illustrates lycopene production in E. coli host cells using a single expression vector (pARC376-Ava 102) containing all three genes.
  • the very active GGPP synthase gene contained in plasmid pARC489D and phytoene synthase gene contained in plasmid pARC140N can be transformed separately or together with the phytoene dehydrogenase- 4H structural gene found in plasmid pARC496A to prepare transformed host E. coli cells that contain all three functional structural genes.
  • expression of plasmids pARC489D and pARC140N provides the enzymes needed to convert ubiquitous cellular precursors into the required phytoene that is converted into lycopene by the action of the phytoene dehydrogenase-4H expressed by plasmid pARC496A.
  • Example 10 illustrates lycopene production in S.
  • cerevisiae host cells transformed with both plasmid pARC145G, whose expression products provides phytoene to the cells, and plasmid pARC146D that expresses phytoene dehydrogenase- 4H that converts the provided phytoene into lycopene.
  • the recombinant DNA molecule preferably contains an expression system that comprises one or more expression vectors compatible with host cells, operatively linked to an exogenous DNA segment that comprises (i) a nucleotide base sequence corresponding to a sequence, defining a structural gene for GGPP synthase, and (ii) a nucleotide base sequence corresponding to a sequence defining a structural gene for phytoene synthase, (iii) a nucleotide base sequence corresponding to the sequence defining a structural gene for phytoene dehydrogenase-4H, and (iv) a nucleotide base sequence corresponding to the sequence defining a structural gene for lycopene cyclase.
  • lycopene is provided to the host cells by the enzymes expressed by the expression system.
  • the structural genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H and lycopene cyclase are contained operatively linked in a single expression vector, preferably under the control of the same promoter.
  • two expression vectors are used, with the structural genes for GGPP synthase, phytoene synthase and phytoene dehydrogenase- 4H on one vector and the structural gene for lycopene cyclase on the other vector.
  • three expression vectors are used. Yeast and plants require a separate promoter for each gene, although the same promoter can be used for each gene.
  • Example 16 illustrates beta-carotene production in E. coli host cells using a single expression vector plasmid pARC376-Pst 102 containing all four genes.
  • the very active GGPP synthase gene contained in plasmid pARC489D, phytoene synthase gene contained in plasmid pARC140N and the phytoene dehydrogenase-4H structural gene found in plasmid pARC496A can be transformed separately or together with the lycopene cyclase structural gene found in plasmid pARC1510 to prepare transformed host E. coli cells that contain all four functional structural genes.
  • expression of plasmids pARC489D, pARC140N and pARC496A provides the enzymes needed to convert ubiquitous cellular precursors into the required phytoene that is converted into lycopene that is subsequently converted into beta-carotene by the action of the lycopene cyclase expressed by plasmid pARC1510.
  • Example 17 illustrates beta- carotene production in plasmid pARC145G, whose expression products provides phytoene to the cells and plasmid pARC1520 that expresses both phytoene dehydrogenase-4H, which converts the provided phytoene into lycopene, and lycopene cyclase that converts lycopene into beta-carotene.
  • the recombinant DNA molecule preferably contains an expression system that comprises one or more expression vectors compatible with host cells, operatively linked to an exogenous DNA segment that comprises (i) a nucleotide base sequence corresponding to a sequence, defining a structural gene for GGPP synthase, and (ii) a nucleotide base sequence corresponding to a sequence defining a structural gene for phytoene synthase, (iii) a nucleotide base sequence corresponding to the sequence defining a structural gene for phytoene dehydrogenase-4H, (iv) a nucleotide base sequence corresponding to the sequence defining a structural gene for lycopene cyclase and (v) a nucleotide base sequence corresponding to the sequence defining a structural gene for beta-carotene hydroxylase.
  • beta-carotene is provided to the host cells by the enzymes expressed by the expression system.
  • the structural genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase and beta- carotene hydroxylase are contained operatively linked in a single expression vector, preferably under the control of the same promoter.
  • two expression vectors are used, with the structural genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H and lycopene cyclase on one vector and the structural gene for beta-carotene hydroxylase on the other vector.
  • three expression vectors are used. Yeast and plants require a separate promoter for each gene, although the same promoter can be used for each gene.
  • Example 1 illustrates zeaxanthin production in E. coli and A. tumefaciens host cells using a single expression plasmid vector pARC288 containing all five genes.
  • Example 21 illustrates zeaxanthin production in E. coli host cells using an expression plasmid vector pARC279 containing all four genes required to produce beta-carotene, but with the gene for beta-carotene hydroxylase having been deleted.
  • E. coli containing plasmid pARC279 were further transformed with the plasmid pARC406BH, and then the cells were grown in appropriate selective medium. Zeaxanthin was produced.
  • the very active GGPP synthase gene contained in plasmid pARC489D can be transformed separately or together with the beta-carotene hydroxylase structural gene found in plasmid pARC406BH to prepare transformed host E. coli cells that contain all five functional structural genes.
  • expression of plasmids pARC489D, pARC140N, pARC496A and pARC1510 provides the enzymes needed to convert ubiquitous cellular precursors into the required phytoene that is converted into lycopene and then beta-carotene, which is subsequently converted into zeaxanthin by the action of the beta-carotene hydroxylase expressed by plasmid pARC406BH.
  • Example 22 illustrates zeaxanthin production in S.
  • plasmid pARC145H cerevisiae host cells transformed with plasmid pARC145H, whose expression products provide GGPP synthase, phytoene synthase and beta-carotene hydroxylase to the cells, and plasmid pARC1520 that expresses both phytoene dehydrogenase-4H and lycopene cyclase.
  • plasmid pARC145H whose expression products provide GGPP synthase, phytoene synthase and beta-carotene hydroxylase to the cells
  • plasmid pARC1520 that expresses both phytoene dehydrogenase-4H and lycopene cyclase.
  • the recombinant DNA molecule preferably contains an expression system that comprises one or more expression vectors compatible with host cells, operatively linked to an exogenous DNA segment, comprising (i) a nucleotide base sequence corresponding to a sequence, defining a structural gene for GGPP synthase, and (ii) a nucleotide base sequence corresponding to a sequence defining a structural gene for phytoene synthase, (iii) a nucleotide base sequence corresponding to the sequence defining a structural gene for phytoene dehydrogenase-4H, (iv) a nucleotide base sequence corresponding to the sequence defining a structural gene for lycopene cyclase, (v) a nucleotide base sequence corresponding to a sequence defining a structural gene for beta-carotene hydroxylase, and a nucleotide base sequence corresponding to a sequence
  • the structural genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta- carotene hydroxylase and zeaxanthin glycosylase are contained operatively linked in a single expression vector, preferably under the control of the same promoter.
  • two expression vectors are used, with the structural genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase and beta-carotene hydroxylase on one vector and the structural gene for zeaxanthin glycosylase on the other vector.
  • Example 1 illustrates zeaxanthin diglucoside production in and recovery from E. coli using a single expression vector pARC376 containing all six genes.
  • Example 26 illustrates zeaxanthin diglucoside production in E. coli host cells using expression vector pARC288 containing all five genes required to produce zeaxanthin plus plasmid pARC2019 with the gene for zeaxanthin glycosylase.
  • E. coli containing pARC288 were further transformed with the plasmid pARC2019, and then the cells were grown in appropriate selective medium. Zeaxanthin diglucoside was produced and recovered.
  • the very active GGPP synthase gene contained in plasmid pARC489D the phytoene synthase gene contained in plasmid pARC140N, the phytoene dehydrogenase-4H gene found in plasmid pARC496A, lycopene cyclase structural gene found in plasmid pARC1510 and the beta-carotene hydroxylase structural gene found in plasmid pARC406GH can be transformed separately or together with the zeaxanthin glycosylase gene found in plasmid pARC2019 to prepare transformed host E. coli cells that contain all six functional structural genes.
  • expression of plasmids pARC489D, pARC140N, pARC496A, pARC1510 and pARC406BH provides the enzymes needed to convert ubiquitous cellular precursors into the required phytoene that is converted into lycopene and then beta-carotene, that is converted into zeaxanthin and then into zeaxanthin diglucoside by the action of the zeaxanthin glycosylase expressed by plasmid pARC406BH.
  • Example 27 illustrates zeaxanthin diglucoside production in S.
  • cerevisiae host cells multiply transformed with plasmid pARC145H, whose expression products provide GGPP synthase, phytoene synthase and beta-carotene hydroxylase to the cells, plasmid pARC1520 that expresses both phytoene dehydrogenase-4H and lycopene cyclase, and a plasmid that expresses zeaxanthin glycosylase.
  • all enzymes required for zeaxanthin diglucoside biosynthesis are found on these three plasmids.
  • the order of expression of the structural genes is not important so, for example, ' the structural gene for GGPP synthase can be located 5' (upstream) from the structural gene for phytoene synthase, or vice versa.
  • this method also contemplates carotenoid production by use of transformed host cells containing only one carotenoid synthesis gene-containing expression vector.
  • the nutrient medium supplies the immediate precursor substrate molecule to the host cells so that those host cells can provide the precursor substrate for the expressed enzyme.
  • the nutrient medium can contain the requisite amount of precursor in micelles or vesicles, as are well known, which are taken up by the host cells.
  • Another aspect of this embodiment contemplates host cells transformed with one, two, three, four or five expression vectors for the production of phytoene synthase, phytoene dehydrogenase-4H, lycopene cyclase, beta-carotene hydroxylase and zeaxanthin glycosylase.
  • GGPP is provided to the transformed host cells via the nutrient medium as above, and the transformed host cells convert the GGPP to the necessary phytoene and then to lycopene, beta-carotene, zeaxanthin and zeaxanthin diglucoside using the transformed structural genes.
  • cells are transformed with fewer than all of the five genes where a carotenoid other than zeaxanthin diglucoside is desired and GGPP is provided by the medium to the cells.
  • the transformed host is free of exogenously supplied DNA segments that inhibit the production and/or accumulation of a desired carotenoid.
  • exogenously supplied DNA that encodes a biologically active zeaxanthin glycosylase that converts zeaxanthin to zeaxanthin diglucoside is absent so that zeaxanthin accumulates.
  • DNA sequencing was performed on M13 single-stranded DNA using a modification of the basic dideoxy method of Sanger et al, Proc. Natl. Acad. Sci. U.S.A. 74:5463-7 (1977). A sequencing kit from BRL Life Technologies, Inc., Gaithersburg, MD was used. The DNA sequence was analyzed on the IG Suite from Intelligenetics Corp. Enzyme assays for enzymes engineered in
  • E. coli or Saccharomyces cerevisiae were performed according to the protocols provided in Example 2e for GGPP synthase and phytoene synthase, in Example 8g for phytoene dehydrogenase-4H, and in Example 15f for lycopene cyclase.
  • Carotenoids were extracted and analyzed by high performance liquid chromatography (HPLC) from both E. coli or S. cerevisiae according to the protocol provided in Example 4.
  • HPLC high performance liquid chromatography
  • the identity of zeaxanthin diglucoside was confirmed by mass spectroscopy performed according to the protocol provided in Example 4.
  • the identity of zeaxanthin was confirmed by mass spectroscopy.
  • the identification of the other carotenoids was confirmed by elution from HPLC, UV-Visible spectral analysis, and comparison with known standards of phytoene, lycopene, and beta-carotene.
  • Example 2d The method for production in E. coli of the proteins in E. coli encoded by the different genes, using the inducible Rec 7 promoter system in the plasmid pARC306A, is described in Example 2d. These proteins were used in the enzyme assays described. This protocol was also used to produce sufficient amounts of the proteins from which the N-terminus of the protein was determined.
  • Examples 21-25 discuss the production of zeaxanthin.
  • Example 21 describes construction of an engineered, readily movable structural gene for beta- carotene hydroxylase
  • Example 22 illustrates the incorporation of that structural gene into plasmid pARC306A to form plasmid pARC404BH, which when placed into E. coli cells along with plasmid pARC279 caused production of zeaxanthin.
  • Example 26 discusses the production of zeaxanthin diglucoside.
  • Example 26 describes construction of an engineered, readily movable structural gene for zeaxanthin glycosylase, which when placed into E. coli along with plasmid pARC288 caused production of zeaxanthin diglucoside. Exa ple l. Confirmation of the presence of the carotenoid biosynthesis pathway genes in Erwinia herbicola plasmid pARC376 a. E. coli
  • E. coli cells which by themselves are not j capable of pigment formation, become intensely yellow in color when transformed with plasmid pARC376 ( Figure 5) .
  • the pigments responsible for the observed yellow color were extracted from the cells and shown to be
  • Plasmid pARC803 (about 17 kb) , which contained the 35 R1162 ori, the kanamycin resistance gene (NPTII) and the Erwinia herbicola DNA of plasmid pARC376- Ava 103 fragment (derived by deleting 2 Ava I restriction fragments, at about 8331-8842-10453, and cloning the Hind III (about 13463) to Eco RI (about 3370 Figure 5) fragment into plasmid pSOC925 ( Figure 12) ;
  • Plasmid pARC274 (about 17 kb) , which contained the R1162 ori, the kanamycin resistance gene, and the Erwinia herbicola DNA of plasmid pARC376-Bam 100 fragment (derived by deleting 2 Bam HI restriction fragments, at about 3442-4487-5302 and cloning the Hind III (about 13463) to Eco RI (about 3370, Figure 5) fragment into plasmid pSOC925;
  • Plasmid pARC288 (about 18 kb) which contained the R1162 ori, the kanamycin resistance gene, the Erwinia herbicola DNA of plasmid pARC376-Sal 8 (Example 2a) and the GGPP synthase gene fragment from Hind III (about 13463) to Eco RV (about 11196, Figure 5) .
  • plasmids were transformed into competent cells of Agrobacterium according to the protocol below. 1.
  • An Agrobacterium colony was grown overnight (about 15 hours) in 2 to 3 ml YP medium (10 g/1 Bactopeptone, 10 g/1 yeast extracts, and 5 g/1 NaCl, pH 7) .
  • the overnight culture was transferred into 50 ml fresh YP medium in 250 ml flask at 250 rpm and
  • the culture was chilled on ice for 5 minutes, then the cells were harvested by centrifugation. 4. The cells were resuspended in 1 ml of 20 mM calcium chloride.
  • reaction mixture was frozen in liquid nitrogen for 1 to 2 minutes and then incubated at 37°C for 5 minutes.
  • the cells were plated in LB medium (5 g/1 yeast extracts, 10 g/1 tryptone, 5 g/1 NaCl, and 2 g/1 glucose, pH 7) containing 50 ⁇ g/ml kanamycin.
  • the transformed cells were selected on LB
  • the carotenoids produced by both E. coli and Agrobacterium are listed in Table 1.
  • the amounts of carotenoids produced by Agrobacterium were about 5 to 10 times lower than by E. coli cells carrying the same
  • the GGPP synthase gene was obtained from the pARC376 plasmid utilizing the following methods. a. Digestion of pARC376 with Sal I
  • the plasmid pARC376-Sal 8 is a derivative of plasmid pARC376 from which two Sal I fragments were removed. One of those fragments is the approximately 1092 bp fragment bounded by the Sal I restriction sites at about 9340 and about 10432 shown in Figure 5, whereas the other is the 3831 bp (approximate size) fragment bounded by the Sal I restriction sites at about 10432 and about 14263 also in Figure 5. This was accomplished as follows.
  • Plasmid pARC376 DNA was prepared using the alkaline lysis method. 5 Micrograms of plasmid DNA were digested with Sal I (BRL) in a high salt buffer provided by the supplier and additionally containing 150 mM NaCl, for 1 hour at 37°C and purified on a 0.8 percent agarose gel. The remaining plasmid, about 10.2 kilobases in length, was electroeluted from the gel, phenol extracted and ethanol precipitated. After elimination of the above Sal I fragments from about positions 9340 to 14263, the remaining DNA was religated to itself to form plasmid pARC376-Sal 8.
  • plasmid pARC376-Sal 8 was cloned into plasmid pSOC925, an E. coli plasmid R1162 derivative, to generate plasmid pARC808.
  • the plasmid pSOC925 contains the origin of replication from the R1162 plasmid, the NPT II gene from Tn5 that confers resistance to kanamycin, and unique Hind III and Eco RI restriction sites.
  • the plasmid pS0C925 expression DNA vector was prepared for cloning by admixing 5 ⁇ g of plasmid DNA to a solution containing 5 units of each of the restriction endonucleases Hind III and Eco RI and the Medium Salt Buffer from Maniatis. This solution was maintained at 37°C for 2 hours. The solution was heated at 65°C to inactivate the restriction endonucleases. The DNA was purified by extracting the solution with a mixture of phenol and chloroform followed by ethanol precipitation.
  • Plasmid pARC376-Sal 8 was digested with Hind III and Eco RI in a similar way.
  • the Erwinia herbicola DNA in plasmid pARC376-Sal 8 from the Hind III site at about position 348 to the Eco RI site at about position 3370 ( Figure 5) was then ligated into the plasmid vector pSOC925 that had already been digested with Hind III and Eco RI.
  • the ligation reaction contained about 0.1 ⁇ g of the plasmid vector pSOC925 and about 0.2 ⁇ g of the Erwinia herbicola Hind III to Eco RI fragment from plasmid pARC376-Sal 8 in a volume of 18 ⁇ l. Two ⁇ l of 10 X ligation buffer (IBI, Corp) and 2 units of T4 ligase were added. The ligation reaction was incubated at 4"C overnight (about 15 hours). The ligated DNA was transformed into E. coli HB 101 according to standard procedures (Maniatis) . This generated the plasmid pARC808, which also codes for kanamycin resistance. The excised DNA fragment from plasmid pARC376-Sal 8 contains an endogenous promoter sequence upstream from the GGPP synthase gene.
  • Positive clones with inserts were identified by growing prospective positive clones, isolating plasmid DNA by the alkali lysis method (Maniatis) , and performing restriction enzyme analysis on the isolated plasmid DNA's. E. coli cells transformed with this plasmid DNA did not produce colored carotenoids, as determined by visual inspection and HPLC and TLC analysis. Other studies discussed hereinafter demonstrated that plasmid pARC808 expresses Erwinia herbicola enzymes that can convert phytoene into colored carotenoid pigments.
  • a second plasmid was constructed by inserting a restriction fragment containing the approximately 1153 bp Bgl II (about position 12349, Figure 5) to Eco RV (about position 11196, Figure 5) fragment from plasmid pARC376 into the Bam HI and Hind III sites of
  • the plasmid pARC273 contains the Erwinia herbicola DNA from the Bgl II site (at about position 12349) to the Eco RV site (at about position 11196) .
  • 25 fragment (about 0.2 mg) from plasmid pARC273 was admixed with the Bam HI and Hind III digested plasmid pBR322 vector (about 0.1 ⁇ g) in 18 ⁇ l total volume.
  • Two ⁇ l of 10 X Ligation Buffer (IBI, Corp.) and 2 units of T 4 Ligase were added, the reaction was incubated
  • This plasmid, pARC282 encodes ampicillin resistance in E. coli and includes a native Erwinia herbicola promoter between the Bgl II site and the initial Met codon of the GGPP synthase gene, but does not cause any carotenoids to be produced. However, when this plasmid was transferred into E. coli cells containing the plasmid pARC808, and the E. coli cells were grown in the presence of both kanamycin and ampicillin, carotenoids were synthesized as evidenced by production of the yellow pigment zeaxanthin.
  • plasmid pARC282 contained the essential gene that was deleted from the plasmid pARC376-Sal 8 plasmid, and the presence of this gene in combination with other Erwinia herbicola carotenoid genes could restore carotenoid production in E. coli.
  • Plasmid Constructs Enzyme assays were performed on similar plasmid constructs, including plasmid pARC491 which was constructed by cloning the approximately 1068 bp fragment from Hpa I (at about position 12264 of plasmid pARC376 or about position 84 of Figure 2) to Eco RV (at about position 11196, Figure 5) into a plasmid denominated pARC306A. Plasmid pARC306A, whose restriction map is illustrated in Figure 6 contains approximately 2519 base pairs. This plasmid contains the polylinker region from pUC18, a unique Nco I site, the ampicillin selectable marker, the pMBl origin of replication and the Rec 7 promoter. Cells containing this plasmid construct had a level of 7.91 nmol/min/mg protein activity of GGPP synthase. e. DNA sequencing
  • the DNA sequence was determined for the approximately 1153 base pair restriction fragment from the region between the Bgl II site at about 12349 of Figure 5 and the Eco RV site at about 11196 of Figure 5.
  • the obtained DNA sequence and putative partial amino acid residue sequences are shown in Figure 2 (about positions 1 to 1153) .
  • the direction of transcription of the gene for GGPP synthase in plasmid pARC376 ( Figure 5) is counterclockwise and proceeds in the direction from the Bgl II site toward the Eco RV site.
  • the initiation codon for GGPP synthase begins at about nucleotide position 12226 of plasmid pARC376 with the ATG codon for methionine (about position 124 of Figure 2) .
  • a Nco I restriction site was introduced at this position of the GGPP synthase gene using in vitro mutagenesis following the techniques described in Current Protocols In Molecular Biology. Ausabel et al. eds., John Wiley & Sons, New York, (1987) p. 8.1.1- 8.1.6, with the exception that E. coli CJ 236 was grown (in step 3 at page 8.1.1) in further presence of 20 ⁇ g/ ⁇ l chloramphenicol.
  • the primer used was:
  • TTG CCATGG GGA (SEQ ID NO:20) wherein a bold-faced letter above and in the following examples indicates an altered base.
  • This modified version of the GGPP synthase gene from the newly introduced Nco I site to the Eco RV site (about 1029 bp) was then inserted into the plasmid pARC306A to generate plasmid pARC417BH.
  • This plasmid, pARC417BH contains the E. coli promoter Rec 7 adjacent to a multiple cloning site. Structural genes lacking a promoter region, when introduced adjacent to the Rec 7 promoter, are expressed in E. coli.
  • GGPP synthase enzyme activity (measured as GGOH) was found at the level of 6.35 nmol/min/mg protein.
  • GGPP synthase enzyme activity measured as GGOH
  • carotenoids were produced. This demonstrated that the gene for GGPP synthase had been identified and genetically engineered.
  • Nco I site (SEQ ID NO:21) 17 amino acids downstream from the initiation codon for the GGPP synthase gene that is located at about position 124 in Figure 2. That site was thus placed at
  • AAG TAATGA GAC (SEQ ID NO:22) was changed to AAG CCATGG GAC.
  • SEQ ID NO:23 This modified GGPP synthase gene coding for seventeen 10 fewer amino-terminal amino acid residues was inserted into plasmid pARC306A at the Nco I site of that plasmid to generate plasmid pARC418BH.
  • Plasmid pARC306A was digested with Eco RI. The Eco RI end was converted to a blunt end using the
  • the resulting partially ligated plasmid was then digested with Hind III, which resulted in the loss of the polylinker region shown in Figure 6 from the Eco RI site to the Hind III site.
  • the resulting Hind III sticky ends were then ligated to form plasmid pARC489B. Positive clones were identified by plasmid DNA isolation (Maniatis) , and by restriction enzyme analysis on the plasmid DNA.
  • plasmid pARC489B When the plasmid pARC489B was transferred to E. coli cells that contained a plasmid containing the 10 rest of the genes coding for enzymes required for carotenoid production, plasmid pARC808, the cells produced carotenoids. Therefore, this construction coded for an active enzyme even though the heterologous gene portion from plasmid pARC306A encoded the first 15 four amino acid residues, and the first 13 amino acid residues encoded by the gene for GGPP synthase were deleted.
  • the above described DNA segment of plasmid pARC489B overlaps bases encoding four amino acids 20 adjacent to the Rec 7 promoter at its 5' end and extends to the blunted, former Eco RI site in the polylinker region of the plasmid.
  • This DNA segment can be excised by reaction with Nco I at its 5' end and the Hind III or Pvu II sites as are illustrated for plasmid 25 pARC306A in Figure 6.
  • the desired GGPP synthase gene does not contain a Pvu II or a Hind III restriction site.
  • the region between the Hind III and Pvu II sites of plasmid pARC489B contains stop codons in all three reading 30 frames. It is preferred to utilize the Pvu II site for ' cleavage of the 3' end of the DNA.
  • GGPP synthase DNA segment can be referred to as lying within the approximately 1150 bp sequence between the Nco I and Pvu II restriction sites of plasmid pARC489B.
  • the 3' end of the gene for GGPP synthase was modified. This construction was made in the following manner. A Nru I (about 11187)-Bal I (about 11347 of Figure 5) double blunt end fragment was inserted into a specially prepared version of the plasmid pARC306A.
  • plasmid pARC306A was digested with Eco RI, and the resulting ends were blunted with the Klenow fragment of DNA Pol I to produce two blunt ends.
  • Nru I-Bal I fragment was operatively linked into the plasmid pARC306A vector to form plasmid pARC489D.
  • This vector included all of the polylinker restriction sites of plasmid pARC306A shown in Figure 6 except the Eco RI site.
  • the GGPP synthase gene-containing portion of the resulting plasmid pARC489D has the same 5* end as does plasmid pARC489B, but the 3' end is about 151 bp shorter than the GGPP synthase gene in plasmid pARC489B.
  • the sequence of the heterologous GGPP synthase structural gene of plasmid pARC489D is illustrated in Figure 3 from about position 150 to about position 1002, with the 5' end of this DNA being the same as that of the GGPP synthase gene present in plasmid pARC489B.
  • GGPP synthase structural gene can be transferred from a pARC306A-derived plasmid such as plasmid pARC489D to other plasmids as an approximately 1000 bp Nco I-Pvu II fragment.
  • Plasmid pARC489D was transformed into E. coli. Very surprisingly, this construction gave the highest enzyme activity of all the different versions of the GGPP synthase gene. This activity was an unexpectedly high 23.28 nmol/min/mg protein.
  • the plasmid pARC489D was introduced into E. coli cells containing the plasmid pARC808, carotenoids were synthesized.
  • the plasmids pARC489B and pARC489D were introduced into the E. coli strain JM101 (BRL) . These cells were treated with nalidixic acid to induce the
  • Rec 7 promoter which caused production of large amounts of the GGPP synthase enzyme.
  • the protein extract from these cells was separated on SDS- polyacrylamide gel electrophoresis (PAGE) . Because of the very large amount of GGPP synthase produced under these conditions, it is readily identifiable by stain- g with Coomassie Brilliant Blue on the SDS-PAGE system. The isolated and substantially purified GGPP synthase can then be recovered from the gels by standard procedures.
  • the Erwinia herbicola GGPP synthase that was produced in cells containing plasmid pARC489B was a protein of the size of about 35 kilodaltons, and is thought to be the complete, native molecule, whereas the GGPP synthase that was produced in cells with plasmid pARC489D was about 33 kilodaltons.
  • the GGPP synthase structural gene present in plasmid pARC489D is the gene most preferably used for GGPP synthase in E. coli, S. cerevisiae, and higher plants.
  • a single colony from a plate containing freshly ( ⁇ 2 days old) transformed cells was picked, grown overnight (e.g. about 15-18 hours) in M9+CAGM medium (see Table 3B hereinafter for media formulations) + 50 ⁇ g/ml ampicillin at 30°C.
  • Cultures of cells were grown at various temperatures from 21-3T C by diluting the cells 1:100 into fresh M9+CAGM medium and maintaining the culture at the desired temperature. Each culture was grown until it was roughly one-half of the final desired density (150-180 Klett units in a shaken culture) .
  • the culture was then induced by addition of nalidixic acid to a final concentration of 50 ⁇ g/ml. Five ⁇ l of a stock solution of freshly prepared 10 mg/ml nalidixic acid in 0.1N NaOH per ml of culture to be induced was used.
  • GGPP synthase was prepared in the cell cytosol as described below.
  • Assay for GGPP synthase Cell cytosol was preincubated for 20 minutes at 4°C with lO ⁇ M epoxy-isopentenyl pyrophosphate (IPP) in order to inhibit IPP-isomerase activity.
  • the assay mixture containing 40 ⁇ M farnesyl pyrophosphate (FPP) and 40 ⁇ M 14C-IPP (250,000 dpm) in 10 mM Hepes buffer (pH 7.0, 1 mM MgCl 2 , 1 mM DTT) in a 1 ml total volume of preincubated cytosol, was incubated at 37 ⁇ C for 30 minutes.
  • the reaction was terminated by transferring the assay mixture to a pre-cooled (in dry ice) tube and lyophilizing for 8 hours.
  • the dry residue was resuspended in 0.5 ml of 0.1 M glycine buffer (pH 10.4, 1 mM MgCl 2 , 1 mM ZnCl 2 ) and treated with 25 units of alkaline phosphatase for 3 hours at 37"C.
  • the alkaline phosphatase reaction converted the pyrophosphates to their corresponding alcohols, which were extracted with hexane, evaporated to dryness under a stream of nitrogen and redissolved in 150 ⁇ l of methanol.
  • Example 3 Phytoene Synthase Gene a. Digestion of pARC376 with Pst I The plasmid pARC376-Pst 122 was created by deletion of an approximately 592 bp Pst I Erwinia herbicola DNA fragment from Pst I sites at about 5807 to about 5215 of plasmid pARC376 ( Figure 5) , followed by religation of the larger of the two fragments.
  • the plasmid pARC305A contains the polycloning linker from pUC18, the chloramphenicol acetyltransferase gene (CAT) that confers chloramphenicol resistance in E. coli and the pMBl origin of replication.
  • the plasmid pARC305A is an analogous plasmid to plasmid pUC18 except plasmid pARC305A contains the CAT selectable marker whereas pUC18 contains the ampicillin selectable marker.
  • plasmid pARC285 used the approximately 1112 bp Nco I to Eco RI fragment from the plasmid pARC376-Bam 100.
  • the plasmid pARC376-Bam 100 is a derivative of the pARC376 plasmid in which the approximately 1045 bp Bam HI fragment from about position 3442 to about position 4482 ( Figure 5) and the approximately 815 bp Bam HI fragment from about position 4487 to about 5302 ( Figure 5) were deleted. A total of about 1860 nucleotides was deleted from the pARC376 plasmid.
  • the Bam HI site at about 5354 at the 3' end was brought within about 72 nucleotides of the Eco RI site originally at about position 3370 of plasmid pARC376.
  • the resulting restriction fragment therefore contained about 1112 bp and was bounded by Nco I and Eco RI restriction sites at its 5* and 3* ends, respectively.
  • the phytoene synthase gene is contained on an approximately 1040 bp Nco I to Bam HI restriction fragment (corresponding approximately to positions 6342 and 5302 of Figure 5, respectively) , but it can be cloned into other plasmids as an approximately 1112 bp Nco I to Eco RI fragment.
  • the approximately 1112 bp Nco I to Eco RI fragment was excised from the plasmid pARC376-Bam 100 and cloned into the Nco I to Eco RI sites of plasmid pARC306A to generate plasmid pARC285.
  • the relevant portion of the phytoene synthase gene can thus be excised from plasmid pARC285 as an approximately 1112 bp Nco I to Eco RI fragment.
  • a Bgl II site was introduced immediately upstream from the methionine codon of the Nco I site, using in vitro mutagenesis, as described before.
  • Two complementary polynucleotide sequences were made that contained a Nco I overhang on one end and on the other end a Bgl II overhang. The sequences were as follows: Bgl II Nco I
  • the two complementary single stranded polynucleotide sequences were hybridized together, ligated to an approximately 1112 bp Nco I-Eco RI fragment from plasmid pARC285 containing the approximately 1040 bp Nco I to Bam HI phytoene synthase gene region and cloned into plasmid pARC135.
  • the plasmid pARC135 (shown in Figure 7) is composed of the pUC18 vector containing the yeast PGK promoter and terminator sequences separated by a unique Bgl II site.
  • the result from the ligation was the following:
  • the plasmid pARC140R contains the S. cerevisiae promoter from the gene for phosphoglyceric acid kinase (PGK) adjacent to the gene for phytoene synthase.
  • PGK phosphoglyceric acid kinase
  • the modified phytoene synthase structural gene was excised from plasmid pARC140R as an approximately 1158 bp Bgl II-Eco RI fragment, engineered and cloned into plasmid pARC306N to generate plasmid pARC140N.
  • the plasmid pARC306N is similar to plasmid pARC306A except that instead of an Nco I site adjacent to the E. coli Rec 7 promoter, there is an Nde I site.
  • Plasmid pARC306N was digested with Nde I and then digested with Sl nuclease to blunt the ends of the former Nde I sites. The plasmid was thereafter digested with Eco RI to remove one of the blunt ends and provide an Eco RI sticky end. Plasmid pARC140R was digested with Bgl II and then with Sl nuclease to blunt the resulting ends. The digested and blunt-ended plasmid was then further digested with Eco RI to remove one of the blunt ends and provide an Eco RI sticky end for the DNA containing the phytoene synthase structural gene. That structural gene was therefore in a fragment of about 1164 bp with a blunt end at one end and an Eco RI site at the other end.
  • the above phytoene synthase structural gene-containing DNA segment was ligated into the blunt end and to Eco RI portions of the above-digested plasmid pARC306N to operatively link the two DNA segments together and form plasmid pARC140N.
  • the phytoene synthase structural gene-containing DNA segment can be excised from plasmid pARC140N as an approximately 1176 bp Hpa I-Eco RI fragment, an approximately 1238 bp Pvu II-Eco RI fragment or as a still larger fragment using one of the restriction sites in the polylinker region downstream from the Eco RI site (see. Figure 6) .
  • the plasmid pARC140N was transferred into E. coli cells that contained the plasmid pARC139, in which part of the gene for phytoene synthase was deleted and, those E. coli cells were therefore incapable of producing any colored carotenoids.
  • plasmid pARC140N was added to those E. coli cells containing plasmid pARC139, the cells produced colored carotenoids. This demonstrated that the modified gene for phytoene synthase coded for a functional enzyme.
  • E. coli cells containing plasmid pARC140N were induced with nalidixic acid to produce large amounts of the phytoene synthase protein according to the protocol discussed hereinbefore.
  • the protein fraction was isolated and analyzed by SDS-PAGE and revealed that the size of phytoene synthase protein is 38 kilodaltons.
  • Example 4 Phytoene Production in E. coli a. Method One - Plasmid containing the engineered genes for GGPP synthase and phytoene synthase
  • a plasmid containing genes for both GGPP synthase and phytoene synthase, as well as an associated promoter regulatory region adjacent to a structural gene causes E. coli cells containing this plasmid to produce phytoene.
  • An example of such a plasmid construct is the use of the structural gene for GGPP synthase from the plasmid pARC489D with a promoter that functions in E. coli adjacent to the 5* end of the structural gene for GGPP synthase. This construct is introduced into a common cloning vector such as pUC18. Where the structural genes are linked together, a single promoter can function in E. coli to express both gene products.
  • a before-described structural gene for phytoene synthase excised from the plasmid pARC140R is cloned adjacent to a promoter that functions in E. coli. such as Rec 7.
  • This Rec 7 promoter-phytoene synthase heterologous gene is then introduced into a plasmid containing the gene for GGPP synthase.
  • the plasmid containing both of these genes directs phytoene synthesis in E. coli.
  • the two genes can also be placed end-to-end in E. coli under the control of a single promoter.
  • Method Two - Plasmid pARC376 with a defective gene for phytoene dehydrogenase- H Phytoene production can occur with the native pARC376 plasmid in which the genes for GGPP synthase and phytoene synthase are functional and produce functional proteins, but in which the gene for phytoene dehydrogenase-4H is impaired, thereby impairing the production of lycopene from phytoene.
  • a plasmid pARC376 derivative in which the gene for phytoene dehydrogenase-4H is deleted or in some other way impaired could not further metabolize the phytoene being produced in the E.
  • phytoene dehydrogenase-4H is located approximately between the positions 7849 to 6380 of plasmid pARC376 as shown in Figure 5.
  • pARC376 derivative plasmids that contain deletions at the beginning of the gene for phytoene dehydrogenase-4H have been made as described before.
  • One plasmid is pARC376-Bam 127, in which the approximately 2749 bp Bam HI fragment from about position 7775 to about 10524 ( Figure 5) was deleted.
  • the other was plasmid pARC376-Pst 110 missing a Pst fragment at 7792-10791 ( Figure 5) .
  • These plasmids were constructed by partially digesting plasmid pARC376 with either Bam HI or Pst I, and ligating the respective DNA fragments together.
  • E. coli cells that contained either plasmid pARC376-Bam 127 or plasmid pARC376-Pst 110 produce phytoene.
  • Phytoene is colorless and cells that produce phytoene have the same colorless character as normal E. coli cells.
  • the ligation mixture was transformed into E. coli and any resulting colorless colonies were analyzed for the presence of phytoene. The presence of phytoene was confirmed by growing E. coli cells containing the plasmid, performing an extraction according to the following protocol, and identifying phytoene by HPLC analysis in the extract.
  • the entire pool of the extract was filtered through a 0.2 micron Acrodisc CR filter in a glass syringe, and the filtrate was dried under nitrogen. Utmost care was taken to protect the carotenoids/ xanthophylls from light and heat.
  • TLC thin layer chromatography
  • the carotenoids/xanthophylls were separated by high pressure liquid chromatography (HPLC) with the aid of a Hewlett Packard C-18 Vydac analytical column (4.6 x 250 mm, 5 micron particle size) .
  • HPLC high pressure liquid chromatography
  • the amount of phytoene produced in these cells averaged about 0.01 percent (dry weight).
  • Example 5 Phytoene Production in S. cerevisiae S. cerevisiae does not normally produce carotenoids since it does not have the necessary functional genes for phytoene production. S. cerevisiae does, however, produce farnesyl pyrophosphate (FPP) .
  • FPP farnesyl pyrophosphate
  • the genes for GGPP synthase and phytoene synthase need to be transferred into the S. cerevisiae cells in the proper orientation to permit the expression of functional enzymes.
  • Promoter sequences that function in S. cerevisiae need to be placed adjacent to the 5 1 end of the structural genes for GGPP synthase and phytoene synthase and termination sequences can also be placed at the 3* ends of the genes.
  • the genes for GGPP synthase and phytoene synthase that contain the proper regulatory sequences for expression in S. cerevisiae then are transferred to the S. cerevisiae cells.
  • 25 plasmid pSOC713 was partially digested with Eco RI and the ends were made blunt with Klenow polymerase and self-ligated.
  • the resultant plasmid contained a unique Eco RI site adjacent to the GAL 1 promoter region. This plasmid was cleaved with Eco RI and the synthetic
  • CAGATCTG GTCTACTG was ligated, cut with Bgl II, and then self-ligated to make a Bgl II site flanked by two Bam HI sites.
  • the restriction map of plasmid pARC145B is shown in Figure
  • the GGPP synthase gene was cloned adjacent to the S. cerevisiae divergent promoter region GAL 10 and GAL 1. so that the GGPP synthase gene would be expressed in S. cerevisiae using the GAL 10 promoter.
  • the gene for phytoene synthase from plasmid pARC140R (Example 2) was excised and placed adjacent to the other side of the GAL 1 promoter of plasmid pARC145F so that the phytoene synthase gene would also be expressed using the GAL 1 promoter.
  • the transcription termination sequence from the S. cerevisiae gene PGK was cloned at the 3' end of the gene for phytoene synthase. More specifically, plasmid pARC145F was digested with Bgl II and Sph I, whose restriction sites are illustrated in Figure 9 for precursor plasmid pARC145B.
  • the phytoene synthase structural gene was excised from plasmid pARC140R as an approximately 1158
  • Plasmid pARC145G The 20 resulting plasmid, now containing both of the genes required for phytoene production under control of the GAL 10 and GAL 1 divergent promoters, was named plasmid pARC145G, and is shown in Figure 10. Other relevant features of plasmid pARC145G include the 2 micron 25 origin of replication of S. cerevisiae and the TRP 1 gene of S. cerevisiae as a selectable marker.
  • the plasmid pARC145G was transferred into the S. cerevisiae strain YPH499 (provided by Dr. Phillip Heiter, Johns Hopkins University) that lacked a 30 functional TRP 1 gene. This strain was able to utilize ** galactose as a carbon source. Transformants were isolated, and the cells were grown in the presence of *• galactose to induce the GAL 10 and GAL 1 promoters to express the genes for phytoene production. The S. cerevisiae cells were grown on the media described below to produce phytoene.
  • YPH499 is a strain of yeast that contains an impaired TRP 1 gene and an impaired URA 3 gene, and is able to utilize galactose as carbon and energy sources.
  • This strain requires tryptophan and uracil in the growth medium in order to grow.
  • these strains can be grown if they are transformed with a plasmid (or plasmids) containing a normal copy of either the TRP 1 gene, but not a normal copy of the URA 3 gene, in which case the cells require uracil to be added to the growth medium, or the URA 3 gene, but not a normal copy of the TRP 1 gene, in which case the cells need to have tryptophan added to the growth medium.
  • plasmid or plasmids
  • Medium 1 is used if the cells contain no further URA 3 or TRP 1 genes.
  • Medium 2 is used if the cells contain a plasmid(s) with only the TRP 1 gene.
  • Medium 3 is used if the cells contain a plasmid(s) with only the URA 3 gene.
  • Medium 4 is used if the cells contain a plasmid(s) with both the TRP 1 and the URA 3 genes.
  • the media constituents are as follows: Basic Constituents:
  • the plasmid pARC145G contains both the GGPP synthase and phytoene synthase genes and a normal copy of the TRP 1 gene. Saccharomvces cells containing pARC145G were grown in Medium 2 with 2 percent galactose.
  • the S. cerevisiae cells were analyzed for the presence of phytoene. A total of 0.12 percent (dry weight) phytoene and related compounds having superimposable UV-Vis spectra as phytoene was found in the cells.
  • the Agrobacterium gene transfer system is preferably used to transfer the genes for GGPP synthase and phytoene synthase to plants.
  • the structural gene for GGPP synthase discussed before is introduced into the plasmid pCaMVCN (Pharmacia) , by replacing the structural gene for chloramphenicol acetyltransferase (CAT) with the gene for GGPP synthase.
  • the Erwinia herbicola GGPP synthase gene is preferably derived from the E. coli plasmid, pARC489D, described above, although another Erwinia herbicola GGPP structural gene can be used.
  • the GGPP synthase gene in pCaMVCN is adjacent to the CaMV 35S promoter, and the NOS polyadenylation site is at the 3' end of the GGPP synthase gene.
  • This gene construct is transferred to the plasmid pGA482 (Pharmacia) .
  • the relevant features of the resulting plasmid are that (i) it contains an origin of replication that permits it to be maintained in Agrobacterium tumefaciens.
  • the gene for phytoene synthase from plasmids pARC140R, pARC140N or another, previously described phytoene synthase structural gene is transferred to pCaMVCN, replacing the CAT gene. This gene is adjacent to the CaMV 35S promoter, and the NOS polyadenylation site is at the 3 1 end of the phytoene synthase gene. This gene is transferred to the pGA482 derivative that already contains the gene for GGPP synthase. The result is a gene construct in which both the genes for GGPP synthase and phytoene synthase can be expressed in plants.
  • This vector is transferred to a suitable vector
  • Agrobacterium tumefaciens strain such as A281 5 (Pharmacia) or LBA4404 (Clontech) . It is noted that * the previously discussed results with A. tumefaciens.
  • Example lb illustrate successful introduction of genes for not only phytoene, but also for additional enzymes in the carotenoid pathway, the successful expression of
  • Suitable plants include tobacco and alfalfa, although others could be used.
  • the genes are expressed in the growing plant, using the CaMV 35S promoter, and the enzymes are deposited in the cytoplasm. Thus, phytoene is produced in the cytoplasm
  • a DNA sequence that encodes a transit peptide sequence, which directs proteins to the chloroplast is introduced in frame at the beginning of the genes for these two enzymes.
  • the 30 order of the gene construction is (i) a promoter that functions in plants, (ii) the DNA sequence for the transit peptide, (iii) the carotenoid structural gene, ' and (iv) a plant polyadenylation sequence.
  • a promoter that functions in plants
  • the DNA sequence for the transit peptide from the "Small Subunit" of the enzyme ribulose bisphosphate carboxylase from tobacco are synthesized from oligonucleotide probes.
  • a Nco I site is placed at the 5' end of the transit peptide sequence, at the initiation methionine codon.
  • a Sph I site is placed at the 3' end of the transit peptide sequence. Details relating to this transit peptide, its gene and the use of its gene are in Example 14.
  • the carotenoid genes for GGPP synthase and phytoene synthase are fused in-frame at the Sph I site of the transit peptide sequence.
  • the chimeric gene is cloned into the plasmid pCaMVCN, replacing the CAT gene.'
  • the result of these constructions is a DNA segment comprising, (i) the plant CaMV 35S promoter adjacent to the transit peptide sequence, followed by (ii) a structural gene for either GGPP synthase or phytoene synthase, or both, and followed by (iii) the NOS polyadenylation site. This gene construct is transferred to the plasmid pGA482.
  • the pGA482 plasmid containing the genes for GGPP synthase and phytoene synthase, is transferred to A. tumefaciens.
  • the genes for GGPP synthase and phytoene synthase are transferred into plants following infection of the plant tissue with the Agrobacterium strain.
  • Suitable host plants include tobacco and alfalfa and others.
  • the genes are expressed from the CaMV 35S promoter, the protein is directed to the chloroplast by the presence of the transit peptide sequence, and the enzyme is delivered inside the chloroplast.
  • the engineered Erwinia herbicola genes for GGPP synthase and phytoene synthase and the ubiquitous precursors, phytoene is accumulated.
  • the gene for phytoene dehydrogenase-4H is found on the plasmid pARC376.
  • the general region of its location on this plasmid was shown by deleting specific regions of the pARC376 plasmid and analyzing the carotenoids produced.
  • the pARC376 plasmid ( Figure 5) was partially digested with either Bam HI or Pst I restriction enzymes, and the free ends were ligated together. This DNA was transformed into E. coli HB101, and colorless colonies were picked and analyzed for the presence of phytoene. Two different plasmid deletions caused the
  • E. coli cells to accumulate phytoene including plasmid pARC376-Bam 127, which had a 2749 bp Bam HI fragment (7745-10524) deletion and plasmid pARC376-Pst 110, which had a 2999 bp Pst I fragment (7792-10791) deletion.
  • the plasmid pARC376-Pst 110 was constructed as follows. Plasmid pARC376 was partially digested with Pst I, the DNA was ligated, the ligation mixture was transformed into E. coli HB101, and the cells were grown in Luria-Broth supplemented with 100 ⁇ g/ml ampicillin. The transformants were screened by isolating plasmid DNA and performing restriction enzyme analysis. A plasmid with only the 2999 bp Pst I segment deleted, was identified and named pARC376-Pst 110. This deletion involves the beginning sequence of the gene for phytoene dehydrogenase-4H.
  • plasmid pARC176B Adjacent to the Eco RI site on the pBluescript plasmid is a Hind III site. There is another Hind III site in the insert from plasmid pARC376 (position 13463) .
  • the plasmid pARC176B was digested with Hind
  • E. coli cells containing plasmid pARC136 were treated with nalidixic acid to induce the Rec 7
  • the 3' end of the phytoene dehydrogenase-4H gene extends beyond the Bgl II site at position 6836
  • the plasmid pARC376 was digested with Sal I restriction enzyme to excise two adjacent DNA segments; an about 1092 bp Sal I segment (positions 9340-10432 of Figure 5) , and an about 3831 bp Sal I segment (positions 10432-14263 of Figure 5) . The free ends of the remaining DNA fragment were religated to form the plasmid, pARC271D.
  • the 3' end of the second strand oligonucleotide probe retaining the native DNA sequence was:
  • Nco I (Nco I site at position 6342 of Figure 5) 5' GG C CAT GG T CTG CGT GGC GTG 3'
  • pARC306A About 100 ng of the Nco I-digested plasmid pARC306A was admixed with about 200 ng of the Nco I fragment produced by the PCR reaction. The fragments were inserted using ligation buffer (2 ⁇ l) (IBI Corp.) and 1 Unit of T4 ligase in a total volume of 20 ⁇ l. The ligation reaction was incubated at 4 ⁇ C for about 15 hours. 4) The ligation mixture was transformed into E. coli HB101. Transformants were selected on Luria-Broth with 100 ⁇ g/ml ampicillin. DNA was isolated from prospective clones and the clone carrying the phytoene dehydrogenase-4H gene insert was identified by restriction enzyme analysis. This plasmid was named pARC496A.
  • the DNA sequence for the phytoene dehydrogenase-4H gene was determined as described before and is shown in Figure 11, along with some of the restriction sites.
  • the approximately 1505 bp Nco I-Nco I fragment (Nco I fragment) present in plasmid pARC496A is a particularly preferred DNA segment herein.
  • kanamycin 5 in the presence of 25 ⁇ g/ml kanamycin.
  • ⁇ E. coli cells containing pARC275 were transformed with the plasmid pARC496A to form doubly transformed host cells. These host cells were grown in medium supplemented with 25 ⁇ g/ml kanamycin and 10 100 ⁇ g/ml of ampicillin. The cells produced lycopene at a level of about 0.01 percent dry weight.
  • the assay for phytoene dehydrogenase-4H was developed using two R. sphaeroides mutants, 1-3 and E-7. 1-3, a mutant strain that has a mutation in the gene for phytoene dehydrogenase-3H, was provided by Dr. Samuel Kaplan, University of Texas Medical Center, Houston, Texas. This mutant, which accumulates phytoene, was used as a source of the substrate for phytoene dehydrogenase-3H and phytoene dehydrogenase-4H.
  • R. sphaeroides E-7 is a strain that cannot make any carotenoids, and was developed at the Amoco Research Center, Naperville, Illinois. This mutant, which has an intact gene for a different, but similar phytoene dehydrogenase-3H, provided a source of the similar enzyme to determine the proper assay conditions.
  • the membrane fraction from the Rhodobacter I- 3 mutant was isolated by growing 1-3 cells until mid to late log phase, pelleting and lysing the harvested cells in 100 mM Tris Buffer, pH 8.0, by vortexing with 150 micron acid-washed glass beads. The cell homogenate was then used as the source of phytoene.
  • the R. sphaeroides E-7 phytoene dehydrogenase-3H transforms phytoene to either phytofluene or neurosporene but not to lycopene, as in Erwinia herbicola.
  • the assay conditions delineated for the Rhodobacter enzyme were also efficacious for the Erwinia herbicola phytoene dehydrogenase-4H.
  • a frozen or fresh cell pellet was resuspended in one volume of 100 mM Tris Buffer, pH 8.0, and lysed by vortexing as described above for Rhodobacter (150 micron beads were used to lyse bacteria and 450 micron beads were used to lyse yeast) .
  • This cell lysate provided a source of phytoene dehydrogenase-4H for testing.
  • GGPP synthase containing the engineered genes for GGPP synthase, phytoene synthase and phytoene dehydrogenase Active GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H enzymes can convert ubiquitous cellular precursors into lycopene.
  • Lycopene was produced in E. coli when plasmids containing the three genes for the above enzymes were introduced into the bacterial host cells.
  • One combination producing lycopene utilized host cells transformed with the plasmids pARC275 and pARC496A.
  • the plasmid pARC275 was constructed in the following manner.
  • the plasmid pARC376-Pst 110 was made by deleting the about 2999 bp Pst I segment (between positions 7792 and 10791, Figure 5) from plasmid pARC376 as described before.
  • the Eco RI (3370) to Hind III (13463 Figure 5) segment from plasmid pARC376-Pst 110 was excised and cloned into the Eco RI to Hind III sites of plasmid pSOC925 to produce plasmid pARC275.
  • the plasmid pSOC925 is about a 9 kilobase plasmid whose restriction map is illustrated in Figure 12.
  • This plasmid contains the kanamycin and chloramphenicol (CAT) resistance genes and the R1162 origin of replication.
  • the chloramphenicol resistance gene can be excised from the plasmid by digestion with Eco RI and Hind III ( Figure 12) .
  • Plasmid pS0C925 was digested with Eco RI and Hind III, excising the CAT gene. About 100 ng of the larger portion of digested plasmid pS0C925 was admixed with about 200 ng of the Eco RI to Hind III fragment from pARC376-Pst 110 in a total volume of 20 ⁇ l to which 2 ⁇ l of Ligation Buffer and 1 Unit of T4 Ligase were added. The ligation mixture was r incubated at 4 ⁇ C for about 15 hours and then 5 transformed into E.
  • Transformants * were grown in .Luria-Broth supplemented with 25 ⁇ g/ml of kanamycin. DNA was isolated from prospective clones and those clones containing the desired DNA insert were identified by restriction analysis. The
  • Transformation of E. coli host cells with plasmids pARC275 and pARC496A produced red colonies of the transformed host cells, as is discussed in
  • the plasmid pARC376-Ava 102 a derivative of plasmid pARC376 in which the gene for lycopene
  • Ava I-Ava I fragments included the about 1633 bp Ava I fragment (10453-8842 Figure 5) and the about 611 bp Ava I-Ava I fragment from (8842- 8231 Figure 5).
  • about 2222 bp of DNA were deleted from the plasmid pARC376.
  • the resulting plasmid pARC376-Ava 102 was transformed into E. coli HBlOl, and the transformants were grown on Luria-Broth with 100 ⁇ g/ml of ampicillin.
  • E. coli cells that contain the entire plasmid pARC376 are yellow due to the production of zeaxanthin and zeaxanthin derivatives. Following transformation, some of the clones were now red in color.
  • GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H on plasmid pARC376-Ava 102 functioned properly and produced lycopene. Because the gene for lycopene cyclase did not function properly, the transformed E. coli host cells accumulated lycopene.
  • Genes sufficient to make lycopene in S. cerevisiae include those for GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H.
  • the plasmid pARC145G (Example 5) has the genes for GGPP synthase and phytoene synthase on both sides and adjacent to the GAL 10 and GAL 1 divergent promoter region. Both of these genes are expressed in S. cerevisiae using these two promoters.
  • the gene for phytoene dehydrogenase-4H is located on the plasmid pARC146D described hereinafter. These two plasmids were transformed into S. cerevisiae, strain YPH499.
  • the yeast strain YPH499 contains a non ⁇ functional TRP 1 gene and a non-functional URA 3 gene (as discussed in Example 5) .
  • Plasmid pARC145G contains a functioning TRP 1 gene as well as the genes for GGPP synthase and phytoene synthase.
  • Plasmid pARC146D contains a functioning URA 3 gene as well as the gene for phytoene dehydrogenase-4H. After both plasmids were introduced, the yeast cells were grown on Medium 4 (Example 5) with galactose to induce the expression of the three carotenoid genes.
  • the cells were grown to stationary phase, collected, extracted, and analyzed by HPLC according to the protocols described before.
  • Yeast cells with the three carotenoid structural genes produced lycopene at about 0.01 percent dry weight.
  • the plasmid pARC146 is a S. cerevisiae vector constructed to direct the expression of the phytoene dehydrogenase-4H gene in yeast.
  • Plasmid pARC145B Figure 9
  • plasmid pARC145B Figure 9
  • Two modifications were made to plasmid pARC145B in order to construct plasmid pARC146.
  • the first modification was the introduction of the PGK terminator at the Sph I site of plasmid pARC145B, downstream from the GAL 1 promoter.
  • a polycloning site, into which a structural gene could be cloned, is present between the GAL 1 promoter and the PGK terminator.
  • I-linkered PGK terminator was ligated to form the resulting plasmid pARC145C.
  • the second modification was to replace the yeast TRP 1 gene with the yeast URA 3 gene.
  • the plasmid pARC145C was digested with restriction enzymes Msc I and Eco RV, and a 737 bp fragment containing the TRP 1 gene was deleted.
  • Synthetic double-stranded sequences containing a potential Xho I cleavage site (BRL) were ligated to the Msc I and Eco RV blunt ends (there are no other Xho I sites in plasmid pARC145) .
  • the resulting DNA fragment was digested with Xho I to produce a DNA having Xho I sticky ends.
  • plasmid pARC146 did not contain two Xho I sites.
  • the apparent loss of the site did not effect the utility of plasmid pARC146 as a URA 3 selectable vector and also did not effect the utility of plasmid pARC146 as an expression vector.
  • this new plasmid construct contains the relevant features of the divergent GAL 1 and GAL 10 promoters, ii) the PGK terminator at the 3' end of the GAL 1 promoter, iii) the 2 micron STB terminator (2 MIC STB TERM) at the 3' end of the GAL 10 promoter, iv) the URA 3 gene that is the selectable marker for transferring the plasmid into S. cerevisiae, and v) the 2 micron origin of replication that permits the maintenance of the plasmid in yeast.
  • This plasmid also contains the pMBl origin of replication for maintenance in E. coli and the ampicillin resistance gene for selection in
  • Plasmid pARC496B was constructed to introduce a Sal I site immediately upstream from the initiation methionine of the phytoene dehydrogenase-4H structural gene and a Sal I site at the 3* end of the gene to enable the gene for phytoene dehydrogenase-4H to be moved as a Sal I-Sal I fragment.
  • This version of the gene was used as the structural gene for phytoene dehydrogenase-4H in constructing the plasmid pARC146D (described below) that was transformed into S. cerevisiae in combination with transformation with plasmid pARC145G to cause the production of lycopene in the transformed yeast.
  • the plasmid pARC496B was constructed using the PCR protocol described before (plasmid pARC496A) to introduce Sal I sites at the 5' and 3' end of the gene.
  • the plasmid pARC271D (Example 8) was digested with Sal I and Xmn I and an about 3035 bp fragment (9340-6305, Figure 5) was isolated after separation on agarose gel electrophoresis. This fragment was used as the template for PCR.
  • the oligonucleotide probe for the 5' end was:
  • the second strand oligonucleotide probe for the 3* end of the gene was:
  • the polymerase chain reaction was carried out as described before. After completion, the reaction mixture was extracted twice with ether and the DNA was precipitated with ethanol.
  • the resulting plasmid was transformed into E. coli HB101, and the transformants were selected by growth in Luria-Broth supplemented with 100 ⁇ g/ml of ampicillin. DNA from prospective clones was isolated and the identity of clones containing the phytoene dehydrogenase-4H gene was confirmed by restriction enzyme analysis.
  • the resultant plasmid was named pARC496B.
  • the about 1508 bp Sal I-Sal I fragment (also referred to as a Sal I fragment) , another particularly preferred DNA segment herein, was cloned from plasmid pARC496B into the yeast vector pARC146, to generate the plasmid pARC146D as described hereinafter.
  • Plasmid vector pATC228 was made by combining the plasmid pATC1619, which contains a genetically engineered phytoene dehydrogenase-4H structural gene, with plasmid pSOC244, which is capable of transforming and being maintained in both E. coli and R. sphaeroides. The following is a description of the multistep construction of plasmid pATC228. a. Construction of Plasmid pATC16l9
  • the plasmid pATC1619 contains a genetically engineered version of the phytoene dehydrogenase-4H gene cloned adjacent to the TAC promoter of pDR540 (Pharmacia) .
  • the gene for phytoene dehydrogenase-4H is expressed in IL. coli and photosynthetic bacteria using the TAC promoter.
  • Plasmid pATC1619 was constructed in a multistep procedure requiring several intermediate plasmids as outlined below.
  • the plasmid pARCBglII401 was constructed by cloning the about 5513 bp Bgl II fragment from plasmid pARC376 (from position 6836 to position 12349 in Figure 5) into the Bam HI site of plasmid pARC306A ( Figure 6) .
  • Plasmid pATC1403 contains a beginning portion of the phytoene dehydrogenase-4H gene.
  • Sph I site was introduced at the initiation MET codon of the phytoene dehydrogenase-4H gene in plasmid pATC1403, using the in vitro mutagenesis protocol described in Current Protocols in Molecular Biology. Ausabel et al. eds., John Wiley & Sons, New York (1987), pp. 8.1.1-8.1.6 (see Example 2).
  • the oligonucleotide probe used as the primer was: Sph I (SEQ ID NO:49)
  • the plasmid, pARC306A ( Figure 6) was digested with Pst I and Sma I.
  • the plasmid pARC376 ( Figure 5) was digested with Pst I and Bal I.
  • An about 1451 bp 30 Pst I (7792) to Bal I (6341) fragment was isolated from an agarose gel. Both, Bal I and Sma I digestions leave a blunt end.
  • the approximately 1451 bp Pst I-Bal I fragment from plasmid pARC376 was cloned into the Pst I and Sma I digested plasmid 35 pARC306A to form plasmid pATC816.
  • Plasmid pARC306A contains an Eco RI site about 30 bp downstream from the Sma I site. The Eco ⁇ . RI site originally present in plasmid pARC306A is maintained in plasmid pATC816. 40 y. Plasmid pATC1605
  • the plasmid pATC1404 contains only the beginning portion of the gene encoding phytoene dehydrogenase-4H. To fuse this portion with the remainder of the phytoene dehydrogenase-4H gene, an about 1052 bp Sma I to Pst I fragment from plasmid pATC1404 (original position 8844 to 7792 of pARC376 in Figure 5) was excised and cloned into plasmid pATC816 (which contains the 3' portion of the phytoene dehydrogenase-4H gene) as follows.
  • Plasmid pATC816 was digested with Ssp I and Pst I (both sites are unique in the pATC816 plasmid) . Digestion with Ssp I left a blunt end. The about 1052 bp Sma I (blunt end) to Pst I fragment from plasmid pATC1404 was cloned into the digested plasmid pATC816, resulting in plasmid pATC1605.
  • the originally present Nco I site shown near the 3' end of the sequence of Figure 11-4 is also present in this construct as is the Eco RI site downstream therefrom that was introduced from plasmid pARC306A.
  • the Sph I-Eco RI fragment of plasmid pATC1605 that contains the structural gene for phytoene dehydrogenase-4H contains about 1550 bp. vi. Plasmid pATC1607
  • Plasmid pATC1605 was digested with Sph I and Eco RI enzymes. The resultant fragment of about 1550 bp was cloned into the plasmid pUC19 (Pharmacia) , which had been digested with Sph I and Eco RI enzymes, resulting in the plasmid, pATC1607.
  • plasmid pATC1607 Upstream and adjacent to the Sph I site on plasmid pATC1607 is a Hind III site that originates from the polylinker region of plasmid pUC19.
  • the structural gene for phytoene dehydrogenase-4H was excised from plasmid pATC1607 by digesting with Hind III and Eco RI. The ends of the resultant fragment, also of about 1550 bp, were blunted by treating with the Klenow fragment of E. coli DNA Polymerase.
  • the plasmid, pDR540 which contains the TAC promoter for gene expression in some bacteria, including E. coli and R. sphaeroides.
  • Plasmid pATC1619 also contains a unique Hind III site.
  • Plasmid pSOC244 is a plasmid that contains i) the R1162 origin of replication, ii) the chloramphenicol acety1transferase gene that confers resistance to chloramphenicol adjacent to the TAC promoter, and iii) a unique Hind III site. This plasmid can transform and be maintained in both E. coli and R. sphaeroides. The construction of plasmid pSOC244 is discussed below.
  • Plasmid PSOC200 Plasmid pQR176a was obtained from Dr. J.A.
  • This plasmid contains the R1162 origin of replication and the transposon Tn5, which confers resistance to kanamycin. This plasmid contains about 14.5 kilobases and contains several
  • Plasmid pQR176a Digestion of plasmid pQR176a with Hind II, followed by religation of appropriate fragments provided plasmid pSOC200, which contained about 8.5 kilobases. This plasmid retained the R1162 origin of replication and the kanamycin resistance gene from
  • Plasmid pSOC200 was digested with Hind III and Sma I endonucleases to remove the kanamycin resistance gene. Plasmid pS0C925 was similarly digested to provide an approximately 1000 bp fragment containing the chloramphenicol acetyltransferase
  • CAT structural gene with the adjacent TAC promoter. That approximately 1000 bp fragment was then cloned into the Hind III- and Sma I-digested plasmid pSOC200 fragment to provide plasmid pSOC244.
  • the resultant plasmid was pATC228, which contains the structural gene for phytoene dehydrogenase-4H and can transform and be maintained in R. sphaeroides. This structural gene can be excised from plasmid pATC228 as an approximately 1506 bp Sph I-Nco I restriction fragment. Plasmid pATC228 is shown schematically in Figure 16.
  • Example 8g possesses an impaired native crtl gene for phytoene dehydrogenase-3H, and thus accumulates phytoene.
  • Cells from R. sphaeroides 1-3 were transformed as hosts with plasmid pATC228. The transformants were selected in the presence of chloramphenicol. The mutant cells that were previously colorless, were colored red after transformation. The red pigment produced by these cells had physicochemical characteristics that were consistent with the properties of the carotenoid spirilloxanthin.
  • the pigment produced by the plasmid pATC228- transformed R. sphaeroides 1-3 mutant host cells was compared to authentic spirilloxanthin extracted from R. rubrum (ATCC 25903) cells grown in culture.
  • the two pigments had the same UV-Vis spectra and the same HPLC profiles.
  • Spirilloxanthin from R. rubrum is derived from lycopene through a series of catalytic steps that include two dehydrogenations, hydration, and then methylation.
  • R. sphaeroides normally transforms phytoene to neurosporene, but not to lycopene, as is the case in Erwinia herbicola. It is believed, therefore, that in the production of the spirilloxanthin-like pigment in the transformed R. sphaeroides. the Erwinia herbicola phytoene dehydrogenase-4H catalyzed desaturation of accumulated phytoene to produce lycopene. The produced lycopene was thereafter further metabolized by native enzymes present in the R. sphaeroides mutant to form spirilloxanthin-like carotenoid.
  • the above-described method is also extendable to other yeasts.
  • One yeast system that serves as an example is the methylotrophic yeast, Pichia pastoris.
  • structural genes for GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H are placed under the control of regulatory sequences that direct expression of structural genes in Pichia. The resultant expression-competent forms of those genes are introduced into Pichia cells.
  • GGPP synthase such as that from plasmid pARC489D is placed downstream from the alcohol oxidase gene (AOX1) promoter and upstream from the transcription terminator sequence of the same A0X1 gene.
  • AOX1 alcohol oxidase gene
  • structural genes for phytoene synthase and phytoene dehydrogenase-4H such as that from plasmids pARC140N and pARC146D are placed between AOX1 promoters and terminators.
  • the vector also contains appropriate portions of a plasmid such as pBR322 to permit growth of the plasmid in E. coli cells.
  • the final resultant plasmid carrying GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H genes, as well as the various additional elements described above, is illustratively transformed into a his4 mutant of P. pastoris. i.e. cells of a strain lacking a functional histidinol dehydrogenase gene.
  • AOX1 promoters After selecting transformant colonies on media lacking histidine, cells are grown on media lacking histidine, but containing methanol as described by Cregg et al. , Molecular and Cellular Biology. 12:3376-3385 (1987), to induce the AOX1 promoters.
  • the induced AOX1 promoters cause expression of the enzymes GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H and the production of lycopene in P. pastoris.
  • the genes encoding GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H as discussed before can be used to synthesize and accumulate lycopene in fungi such as Aspergillus nidulans.
  • the structural gene for GGPP synthase is introduced into the E. coli plasmid pBR322.
  • the promoter from a cloned Aspergillus gene such as argB [Upshall et al., Mol. Gen. Genet. 204:349-354 (1986)] is placed into the plasmid adjacent to the GGPP synthase structural gene.
  • the GGPP synthase gene is now under the control of the Aspergillus argB promoter.
  • the entire cloned amds gene [Corrick et al.. Gene 53:63-71 (1987)] is introduced into the plasmid.
  • the presence of the amds gene permits acetamide to be used as a sole carbon or nitrogen source, thus providing a means for selecting those Aspergillus cells that have become stably transformed with the amds-containing plasmid.
  • the plasmid so prepared contains the Aspergillus argB promoter fused to the GGPP synthase gene and the amds gene present for selection of Aspergillus transformants. Aspergillus is then transformed with this plasmid according to the method of Ballance et al., Biochem. Biophys. Res. Commun. 112:284-289 (1983).
  • the GGPP synthase, phytoene synthase and phytoene dehydrogenase-4H structural genes are each similarly introduced into the E. coli plasmid pBR322. Promoters for the cloned Aspergillus argB gene [Upshall et al., Mol. Gen. Genet. 204:349-354 (1986)] are placed immediately adjacent to the phytoene synthase and phytoene dehydrogenase-4H structural genes. Thus, these structural genes are controlled by the Aspergillus argB promoters.
  • the entire, cloned Aspergillus trpC gene [Hamer and Timberlake, Mol. Cell. Biol., 7:2352-2359 (1987)] is introduced into the plasmid.
  • the trpC gene permits selection of the integrated plasmid by virtue of permitting transformed trpC mutant Aspergillus cells to now grow in the absence of tryptophan.
  • the Aspergillus strain, already transformed with the plasmid containing the GGPP synthase gene, is now capable of synthesizing lycopene.
  • the gene for phytoene dehydrogenase-4H was modified to introduce the restriction site Sph I at the initiation methionine codon, as discussed before.
  • This modified gene was inserted into the plasmid pCaMVCN (Pharmacia, Piscataway, N.J.) replacing the CAT gene.
  • the resultant plasmid contained a gene for phytoene dehydrogenase-4H with the transit peptide sequence placed between and adjacent to both the CaMV 35S plant promoter and the NOS polyadenylation sequence at the 3' end.
  • This phytoene dehydrogenase-4H gene construct was inserted into the plasmid pGA482 (Pharmacia) in a convenient restriction site within the multiple cloning linker region to form plasmid pATC1616.
  • the relevant features of plasmid pGA482 include (i) an origin of replication that permits maintenance of the plasmid in Agrobacterium tumefaciens. (ii) the left and right border sequences from the T-DNA region that direct the integration of the DNA segment between the borders into the plant genome, and (iii) the NOS promoter adjacent to the kanamycin resistance gene that permits plant cells to survive in the presence of kanamycin.
  • This phytoene dehydrogenase-4H gene construct was transformed into Agrobacterium tumefaciens LBA4404 (Clontech, Inc.) according to standard protocols.
  • Agrobacterium cells containing the plasmid with the phytoene dehydrogenase-4H gene construct were transferred by infection of tobacco leaf discs using the method of Horsch et al.. Science. 227:1229-1231 (1985).
  • the entire DNA segment between the left and right borders of the plasmid pGA482 plasmid is transfected into the plant cells. Transfected plant cells are selected for kanamycin resistance.
  • the sequence of the transit peptide DNA 25 is basically that of Mazur et al., Nucl. Acids Res.. .13.:2343-2386 (1985) for the ribulose bis-phosphate carboxylase-oxygenase signal peptide of Nicotiana tabacum.
  • Two changes were made to the disclosed 177 bp sequence. 30 In the first change, two cytidine residues were added at the 5' end to create a Nco I restriction site. The second change introduced an Nar I site that cleaves between bases at positive 73 and 74. This change was a G for T replacement at 35 position 69 and a G for A replacement at position 72, both of which changes left the encoded amino acid residue sequence unchanged.
  • the DNA encoding the transit peptide was synthesized synthetically from eight fragments that were annealed together in pairs by heating at 90°C for five minutes and then slowly cooling to room temperature. Fifty picomoles of each fragment were utilized. Those eight fragments were:
  • Fragment 1-2 was ligated with fragment 3-4 to
  • Fragment 5-6 was ligated with fragment 7-8 to form fragment 5-8 whose sequence is shown below.
  • fragments 1-4 were ligated together over a two hour time period, as were pairs 5-6 and 7-8 to form two double-stranded sequences.
  • the ligation product of fragments 1-4 was digested with Nco I and Nar I, whereas the product of fragments 5-8 was digested with Nar I and Sph I. These digestions separated any concatamers formed during ligation and provided the necessary sticky ends for further ligation.
  • the digested mixes were run on 6 percent acrylamide gels.
  • the bands of correct size were excised from the gels, and the DNA was eluted from the gel matrix.
  • the DNA fragments of (1-4) and (5-6) were ligated together to form a 177 base pair molecule.
  • the ligation was digested with restriction enzymes to create the necessary ends for subsequent cloning of the molecule.
  • the ligation of fragments (1-4) and (5-8) was digested with Nco I and Sph I.
  • the digested ligation product DNA segment was run on a 6 percent polyacrylamide gel. The band of 177 base pairs was excised and eluted from the gel.
  • Plasmid pARC466 is a plasmid identical to M13mpl9 except that an Nco I site has replaced the native Hind III site. This plasmid contains a polylinker region including a Sma I site that is downstream from the Sph I site.
  • the primer used was:
  • Plasmid pARC466 was digested with Nco I and Sph I. The 177 bp transit peptide DNA fragment ends
  • Plasmid pARC480 was sequenced by M13 protocol to check the sequence of the designed peptide, which sequence was found to
  • pCaMVCN is a plasmid supplied by Pharmacia that contains the 35S promoter and a NOS polyadenylation sequence.
  • the transit peptide was cloned next to the 35S promoter as follows: a) Plasmid pCaMVCN was digested with the 30 restriction enzyme Sal I. Linker #1104 from New
  • Plasmid pATC209 was digested with 35 Sma I. Plasmid pARC480 was digested with Nco I and
  • Plasmid pATC1616 is a derivative of plasmid pGA482 that contains the gene for phytoene dehydrogenase-4H with the transit peptide sequence in frame with the coding sequence of the phytoene dehydrogenase-4H gene. This gene construct is driven by the CaMV 35S promoter and contains the NOS polyadenylation site downstream of the structural gene. The plasmid was made in the following way.
  • the plasmid pATC1607 contains a version of the phytoene dehydrogenase-4H with a Sph I site at the initiation methionine codon. Plasmid pATC1607 was digested with Nco I. The cleaved Nco I site is the same as the Nco I site at position 6342 in Figure 5 and is the Nco I site at about position 1510 in Figure 11. The Nco I site was made blunt by treating with the Klenow fragment of DNA polymerase.
  • the thus treated plasmid pATC1607 plasmid was then digested with Sph I. This digestion caused the production of about 1506 bp fragment, which includes the structural gene for phytoene dehydrogenase-4H. At the 5' end of the fragment is a Sph I site and at the 3' end of the fragment is a blunt end.
  • Plasmid pATC212 was digested with Sph I and Sma I.
  • The.Sph I site is at the 3' end of the transit peptide sequence and the Sma I site is downstream in the polylinker sequence of the plasmid pATC212.
  • the above Sph I-blunt ended phytoene dehydrogenase-4H gene fragment was cloned into the pATC212 plasmid, resulting in plasmid pATC1612.
  • Plasmid pATC1612 contains the CaMV 35S
  • This whole region of pATC1612 can be moved as an Xba I-Xba I fragment, since there are Xba I sites upstream from the CaMV
  • Plasmid pATC1612 was digested with Xba I and the about 2450 bp Xba I-Xba I fragment (450 bp CaMV 35S promoter, 177 bp transit peptide sequence, 1506
  • the carotenoid genes described before are introduced into appropriate vector(s) , as also described above for chloroplasts, using identical techniques, except that
  • the transit peptide is eliminated. Because they are not targeted to the chloroplast, the enzymes remain in the cytoplasm, and, acting on the ubiquitous isoprenoid intermediate, farnesyl pyrophosphate, produce lycopene in the cytosol.
  • lycopene cyclase 35 other enzyme genes. If the gene for lycopene cyclase were deleted, mutated or otherwise impaired, there would not be an active lycopene cyclase enzyme and lycopene would accumulate. Lycopene imparts a red color to E. coli cells producing it, whereas beta- carotene imparts a yellow color to E. coli cells producing beta-carotene.
  • Plasmid pARC376 was partially digested with Ava I, the ends were religated, and the plasmid DNA was transformed into E. coli cells strain HBlOl.
  • This plasmid named pARC376-Ava 102, contained a 611 bp Ava I fragment deletion from position 8231 to 8842 and also a 1611 bp Ava I fragment deletion from position 8842 to 10453.
  • Example 8b describes the construction of plasmid pARC137B, whose Erwinia herbicola DNA insert is diagrammatically illustrated below.
  • the resulting plasmid also contained two Stu
  • Plasmid pARC1009 was transformed into E. coli. strain JM101, and the cells were grown and treated with nalidixic acid to induce the Rec 7
  • the protein fraction was isolated, analyzed on PAGE and a dominant protein band of 36 kilodaltons was noted. This protein band was identified as the enzyme lycopene cyclase, as discussed hereinafter. The protein band was isolated
  • 35 sequence of the pARC376 plasmid revealed the position of the initiation codon of the lycopene cyclase gene.
  • the initiation codon is GTG, not the much more common ATG.
  • a GTG codon normally codes for the amino acid valine, but under rare instances in bacteria, it can also code for methionine when it is the first amino acid in a protein (G.D. Stormo, 1986, in Maximizing Gene Expression, W. Reznikoff, L.
  • Plasmid pARC 65 A series of studies was performed to determine the location of the 3' end of the gene. A plasmid, pARC465, which contains the carotenoid genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H and the chloramphenicol acetyltransferase gene that confers resistance to the antibiotic chloramphenicol, was constructed as follows.
  • the plasmid pARC307D is an analogous plasmid to the plasmid pUC8, except that plasmid pARC307D contains the chloramphenicol acetyltransferase gene instead of the ampicillinase gene. Plasmid pARC307D also contains the same polycloning linker as pUC8.
  • Plasmid pARC307D was digested with Hind III and Eco RI.
  • the plasmid pARC376-Ava 102 (Example 9b) was also digested with Hind III and Eco RI.
  • the resultant about 8000 bp fragment from Hind III (13463) to Eco RI (3370) of plasmid pARC376-Ava 102 was isolated from an agarose gel (the fragment size is only about 8000 bp because the Ava I deletions in plasmid pARC376-Ava 102 described before deleted about 2200 bp from the parent pARC376 plasmid) .
  • This about 8000 bp Hind III-Eco RI fragment was cloned into the Hind III- and Eco Rl-digested plasmid * pARC307D.
  • the plasmid pARC1009 which contains the gene for lycopene cyclase, was introduced into E. coli 10 cells containing plasmid pARC465, and the cells were grown on chloramphenicol and ampicillin. These cells produced beta-carotene. This indicated that the 3' end of the gene for lycopene cyclase was upstream from the Stu I site (original position about 7306) . 15 d. Plasmid pARClOO ⁇
  • the 1548 bp Sal I (9340) to Pst I (7792) DNA fragment was cloned into plasmid 20 pARC306A.
  • the resulting plasmid, pARC1008, was introduced into E. coli cells that already contained plasmid pARC465. These cells, grown in the presence of chloramphenicol and ampicillin, produced beta- carotene. These results indicated that the 3' end of 25 the gene was present upstream from the Pst I (7792) site.
  • the gene for lycopene cyclase is contained in an about 1548 bp Sal I to Pst I fragment of plasmid pARC376.
  • the actual initiation 30 codon is about 338 bp downstream from the Sal I site.
  • the bounds of the gene for lycopene cyclase are approximately from position 9002 to the ** Pst I site at position 7792 in Figure 5, enclosing an approximately 1210 bp DNA segment.
  • Figure 19 35 contains a nucleotide sequence obtained from single strand sequencing and a partial amino acid sequence obtained by amino acid sequencing for lycopene cyclase.
  • the initiation codon was changed from a GTG sequence to an ATG sequence by introducing a Nco I site by in vitro mutagenesis at the beginning of the gene as follows.
  • An oligonucleotide probe was synthesized that had the following sequence as compared with the normal sequence:
  • the Nco I restriction site sequence is CC ATGG, therefore, the new sequence at the initiation methionine introduced an Nco I site.
  • This newly modified lycopene cyclase gene, starting at the introduced Nco I site was cloned into the plasmid pARC306A to generate the plasmid pARC147.
  • Plasmid pARC147 was introduced into E. coli cells already containing plasmid pARC465, and the cells were grown in the presence of chloramphenicol and ampicillin. These cells produced beta-carotene.
  • the assay mixture was thereafter lyophilized and extracted with acetone:methanol (7:2, v:v) .
  • the assay mixture was thereafter lyophilized and extracted with acetone:methanol (7:2, v:v) .
  • Cofactors such as FAD, NADP and FMN are not required for lycopene cyclase activity. ATP is, however, essential for activity.
  • Plasmid pARC1606 The construction of plasmid pARC1606 proceeded with a series of intermediate vectors.
  • the plasmid pARC376 was partially digested with Bam HI and then religated.
  • the religated 35 plasmid was transformed into E. coli cells and cells were selected that contained a plasmid in which Bam HI fragments of about 1045 bp (from original position 3442 to 4487) and of about 815 bp (from original position 5302 to 4487) were deleted from the pARC376 plasmid.
  • the name of the new plasmid was pARC376-Bam 100, and the plasmid caused the E. coli cells to produce ,9-carotene, since the gene for ⁇ -carotene hydroxylase was deleted.
  • the plasmid pARC376-Bam 100 was digested with Hind III and Eco RI. The fragment containing the
  • Plasmid pARC307D contains the pUC8 MCS. Plasmid pARC307D was digested with Hind III and Eco RI, and the Erwinia herbicola Hind III and Eco RI fragment excised from plasmid pARC376-Bam 100 was cloned into plasmid pARC307D to form plasmid pARC279. This plasmid conferred chloramphenicol resistance to the E. Coli cells and also caused them to produce ⁇ -carotene. The plasmid pARC279 contains about 11.7 kb.
  • Plasmid pARC279 was partially digested with Bgl II and Bam HI and then religated to delete specific regions from the pARC279 plasmid that were not necessary for ⁇ -carotene production and make the plasmid as small as possible.
  • a clone was found in which the size of the plasmid was about 10 kb (about 1.7 kb had been deleted), that conferred chloramphenicol resistance to E. coli and caused the synthesis of ,9-carotene. That plasmid was named pARC281B.
  • Plasmid pARC1606 was made from plasmid pARC281B by mutagenizing E. coli cells that contained plasmid pARC281B with nitrosoguanidine (NTG) according to the following protocol.
  • the cells were washed twice with phosphate buffer (50 mM, pH 7.0), and
  • NTG was added to the cells in phosphate buffer to a final concentration of 100 ⁇ g/ml. The cells were incubated for 1
  • the cells were then diluted and plated on Luria-Broth Medium with 1.5 percent Agar with 25 ⁇ g/ml chloramphenicol. A colony was found that produced lycopene as
  • plasmid PARC1606 A mutation was induced somewhere in the gene 30 for lycopene cyclase after the nitrosoguanidine treatment that caused the inactivation of the enzyme. This caused the cells to accumulate lycopene, the ** precursor to ,3-carotene. Cells that contained the plasmid with this mutation were now red, due to the accumulation of lycopene, instead of the ?-carotene yellow color.
  • Cells containing plasmid pARC1606 were used as a source of lycopene for the lycopene cyclase assays described before.
  • Met codon and a Bam HI restriction site at the 3' end of the gene were introduced into the native seguence by PCR (as described before) using the following probes:
  • the resulting plasmid was called pARC1509.
  • pARC1509 in the polycloning seguence is a unique Hind III site.
  • the plasmid pARC1509 was digested with Hind III and Bam HI, an about 1156 bp Hind III- Ba HI fragment was isolated. The fragment ends were made blunt by treatment with the Klenow fragment of
  • Plasmid pKK223-3 contains a unique Eco RI site adjacent to the TAC promoter. Plasmid pKK223-3 was digested with Eco RI and the ends were likewise blunted with the Klenow reagent. The fragment
  • ** pARC1510 was introduced into E. coli cells that already contained the plasmid pARC465 that contains the CAT resistance gene and the genes necessary to produce lycopene, but from which the gene for
  • Beta-carotene production in E. coli a Method One - Plasmid(s) containing engineered genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase- H and lycopene cyclase
  • Four carotenoid enzyme genes are required to produce beta-carotene from ubiquitous precursors, i.e., the genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, and lycopene cyclase.
  • the first three genes i.e., for GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H enzymes, were present on the plasmid pARC465.
  • This plasmid also contains the chloramphenicol acetyltransferase gene that confers resistance to the antibiotic chloramphenicol in E. coli.
  • the plasmid pARC1009 contains the about 2038 bp Sal I to Stu I DNA fragment inserted into plasmid pARC306A.
  • plasmid pARC1009 was transferred to E. coli cells that contained the plasmid pARC465, the cells produced beta-carotene at a level of about 0.05 percent (dry weight) .
  • the plasmid pARC147 also described in Example 15, contains the about 1215 bp Nco I to Pst I fragment that was inserted into the pARC306A plasmid.
  • This plasmid was also introduced into E. coli cells that contained the plasmid pARC465, and those cells also synthesized beta-carotene at a level of about 0.05 percent (dry weight). Because it was subsequently discovered that this version of the lycopene cyclase structural gene was inactive in yeast, its use was discontinued and the gene was altered as described in Example 15 to produce plasmid
  • the plasmid pARC376 has a sufficient gene complement to effectuate the synthesis of carotenoids up to and including zeaxanthin diglucoside in E. coli.
  • Beta-carotene is the metabolic substrate for the beta-carotene hydroxylase enzyme that adds
  • beta-carotene hydroxylase 15 two hydroxyl groups at the 3 and 3' positions of beta-carotene to produce zeaxanthin. If the gene for beta-carotene hydroxylase is deleted, mutated, or in some other way made non-functional, the cells accumulate the substrate beta-carotene.
  • Plasmid pARC376-Pst 102 The gene for beta-carotene hydroxylase is contained on a 975 bp DNA fragment bounded by a Pst I site (4886) and the Sma I site (5861) in plasmid
  • plasmid pARC376 was partially digested with Pst I, and the appropriate cut ends were religated. Analysis of the plasmid DNA determined that the 392 bp Pst I fragment from original position 4886 to 5215
  • This plasmid was named pARC376-Pst 102.
  • Plasmid pARC376-Bam 100 In an analogous procedure, plasmid pARC376 was partially digested with Bam HI and appropriately religated, causing the deletion of an approximately 815 bp fragment from about original position 4487 to 5302. The resultant plasmid was called pARC376-Bam 100. The plasmid DNA was transformed into E. coli HBlOl, and orange-yellow colonies were selected and analyzed for carotenoid content. Beta-carotene accumulated in these cells at a level of about 0.1 percent.
  • the structural gene for each of the four enzymes required for beta-carotene synthesis is placed adjacent to an appropriate promoter and termination sequence that will properly function in S. cerevisiae.
  • Appropriate promoters include the GAL JL and GAL 10 divergent promoters, described in the Detailed Description and Example 5, and the phosphoglyceric acid kinase gene promoter (PGK) , likewise described.
  • An appropriate terminator is the termination sequence from the PGK gene.
  • the structural genes for GGPP synthase and phytoene synthase are present in the plasmid PARC145G, adjacent to the GAL 10 and GAL 1 promoters as described in Example 5.
  • the termination sequence from the PGK gene is at the 3' end of the gene for phytoene synthase.
  • One approach to induce beta-carotene synthesis in yeast is to insert these two genes into a vector, such as pARC146, that contains the GAL 10 and GAL 1 divergent promoters and introduce the 10 resultant plasmid into S. cerevisiae that already contains plasmid pARC145G.
  • the resulting population has all of the genetic material required to produce beta-carotene in a form that permits high level expression of the genes.
  • Plasmid pARC1520 Plasmid pARC1520
  • the plasmid pARC146D (Example 10) already contains the gene for phytoene dehydrogenase-4H adjacent to the GAL 1 promoter.
  • the structural gene 20 for lycopene cyclase described in Example 15 was cloned into plasmid pARC146D adjacent to the GAL 10 promoter as follows:
  • the plasmid pARC1509, described in Example 15, was digested with Hind III and Bam HI.
  • the about 25 1156 bp fragment containing the structural gene for lycopene cyclase was isolated and the ends were blunted by treatment with the Klenow fragment of DNA Polymerase I.
  • Plasmid pARC146D was digested with Eco RI 30 (restriction site is unique in plasmid pARC146D - see
  • Plasmid pARC1520 contains the 35 gene for phytoene dehydrogenase-4H adjacent to the GAL 1 promoter, the gene for lycopene cyclase adjacent to the GAL 10 promoter, and the URA 3 gene (described before) useful for selection in yeast. Plasmid pARC1520 was introduced into the S_j. cerevisiae. strain yPH499, which already contained the plasmid pARC145G. Beta-carotene was produced at the level of about 0.01 percent of the dry weight.
  • beta-carotene is synthesized in the chloroplasts of plants, most higher plant species do not accumulate very high levels of it. Carrot roots are among the best accumulators, but even in these the concentration is only about 0.01-0.1 percent (dry weight) .
  • the objective is to increase the catalytic activity of lycopene cyclase and thereby the accumulation of beta-carotene. Lycopene production is thought to be the divergence point of carotenoid synthesis. In one branch, lycopene is converted to alpha-carotene that in turn is converted to lutein. Lutein is the carotenoid that accumulates to the highest concentration level of all carotenoids.
  • lycopene is converted to beta-carotene, which does not accumulate to as high a level as the lutein. If the level for the enzyme for lycopene cyclase is increased, however, beta-carotene accumulates to higher levels.
  • the resulting plasmid is cleaved with Xba I, and the Xba I-Xba I fragment containing the CaMv 353 promoter (about 450 bp) , the transit peptide (about
  • pGA482 The relevant features of pGA482 were described in Example 14 and include (i) the left and right borders of the T-DNA sequence, which directs the integration of the DNA sequences between these borders into the plant genome; (ii) the kanamycin
  • pGA482 was introduced into Agrobacterium tumefaciens. strain A281.
  • genes for GGPP synthase, phytoene synthase, and phytoene dehydrogenase-4H are examples of carotenoid enzyme-specific genes.
  • the introduction of these genes into higher plants involves the same manipulations as described above for lycopene cyclase.
  • the genes are attached to the tobacco transit peptide DNA sequence and are then placed adjacent to a functional plant promoter, such as the CaMV 35S promoter. Also placed adjacent, is a polyadenylation sequence, such as the NOS polyadenylation sequence.
  • the structural genes are individually cloned into one or more vectors that contain a promoter and a polyadenylation seguence that will function in plants.
  • One such vector is the before-described pCaMVCN, with the CaMV 35S promoter and the NOS polyadenylation sequence.
  • the four genes with the appropriate promoters and polyadenylation signals are then inserted into the before-described plasmid, pGA482. Plasmid pGA482, containing the four carotenoid-specific genes with the appropriate regulatory signals, is transformed into A ⁇ .
  • the before-described method is also extendable to other yeasts.
  • One yeast system that serves as an example is the methylotrophic yeast, Pichia pastoris.
  • GGPP synthase such as that from plasmid pARC489D is placed downstream from the alcohol oxidase gene (AOX1) promoter and upstream from the transcription terminator sequence of the
  • the vector also contains appropriate portions of a plasmid such as pBR322 to permit growth of the plasmid in E. coli cells.
  • a plasmid such as pBR322 to permit growth of the plasmid in E. coli cells.
  • the final resultant plasmid carrying GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H and lycopene cyclase genes, as well as the various additional elements described above, is illustratively transformed into a his4 mutant of P. pastoris, i.e. cells of a strain lacking a functional histidinol dehydrogenase gene.
  • AOX1 promoters After selecting transformant colonies on media lacking histidine, cells are grown on media lacking histidine, but containing methanol as described by Cregg et al. , Molecular and Cellular Biology. 12:3376-3385 (1987), to induce the AOX1 promoters.
  • the induced AOX1 promoters cause expression of the enzymes GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H and lycopene cyclase and the production of ⁇ -carotene in P. pastoris.
  • the four genes for GGPP synthase, phytoene synthase, phytoene dehydrogenase-4H, and lycopene cyclase can also be introduced by integrative transformation, which does not require the use of an ARS sequence, as described by Cregg et al.. Molecular and Cellular Biology. 12:3376-3385 (1987).
  • the structural gene for GGPP synthase is introduced into the. E. coli plasmid pBR322.
  • the plasmid so prepared contains the Aspergillus argB promoter fused to the GGPP synthase gene and the amds gene present for selection of Aspergillus transformants. Aspergillus is then
  • 35 structural genes are each similarly introduced into the E. coli plasmid pBR322. Promoters for the cloned Aspergillus argB gene [Upshall et al., Mol. Gen. Genet, 204:349-354 (1986)] are placed immediately adjacent to those three structural genes. Thus, these structural genes are controlled by the Aspergillus argB promoters.
  • the entire, cloned Aspergillus trpC gene [Hamer and Timberlake, Mol. Cell. Biol.. 7:2352-2359 (1987)] is introduced into the plasmid.
  • the trpC gene permits selection of the integrated plasmid by virtue of permitting transformed trpC mutant Aspergillus cells to now grow in the absence of tryptophan.
  • the Aspergillus strain, already transformed with the plasmid containing the GGPP synthase gene, is now capable of synthesizing ⁇ -carotene.
  • Example 21 Production of Zeaxanthin in E. coli a. Construction of Plasmid pARC404BH The about 2938 bp fragment from the Eco RV site at position 4323 to the Stu I site at position 7306 from plasmid pARC376 ( Figure 5) was cloned into the Sma I site of M13mpl9 (obtained from BRL) . The resulting plasmid was named pARC404BH-B. This plasmid was used for the introduction of an Nco I site at the initiation methionine of the ⁇ -carotene hydroxylase enzyme (position 4991 of Figure 5) using the method described in Ausabel et al. and discussed in Example 2(f) . The oligonucleotide probe used for the in vitro mutagenesis to introduce the Nco I site was:
  • the plasmid with the newly introduced Nco I site at the initiation methionine residue was named plasmid pARC404BH-C.
  • Plasmid pARC404BH-C was digested with Nco
  • This plasmid was called pARC404BH-A.
  • Plasmid pARC376 was digested with Bam HI and Sma I, and the about 559 bp fragment from Bam HI (5302 of Figure 5) to Sma I (5861 of Figure 5) was isolated.
  • the plasmid pARC404BH-A was digested with
  • 35 contains the structural gene for 0-carotene hydroxylase with the newly introduced Nco I site at the beginning of the gene, whose sequence is included in Figure 21.
  • the structural gene can be moved as an about 870 bp Nco I-Sma I fragment.
  • Plasmid pARC404BH was digested with Nco I and Sma I, and the about 870 bp Nco I-Sma I fragment was isolated.
  • the about 870 bp Nco I-Sma I fragment was cloned into the pARC306A plasmid to form to form plasmid pARC406BH.
  • This plasmid contains the structural gene for ⁇ -carotene hydroxylase adjacent to the E. coli Rec 7 promoter.

Abstract

Segments ADN codant les enzymes Erwinia herbicola la syntase geranylgeranyle pyrophosphate (GGPP), la syntase phytoène, la phytoène déshydrogénase-4H, lycopène cyclase, beta-carotène hydroxylase, et zéaxanthine glycocylase et leurs variantes ADN codant une enzyme ayant sensiblement la même activité biologique, vecteurs contenant ces segments ADN, cellules hôtes contenant les vecteurs et procédés de production de ces enzymes, procédé de protection des plantes contre le norflurazon herbicide, ainsi que procédé de production de GGPP et les caroténoïdes phytoène, lycopène, β-carotène, zéaxanthine et zéaxanthine diglucoside par une technologie d'ADN de recombinaison dans des organismes hôtes transformés.
PCT/US1991/001458 1990-03-02 1991-03-04 Biosynthese de carotenoïdes dans hotes transformes par genetique WO1991013078A1 (fr)

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US5656472A (en) 1997-08-12
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